Integration site-dependent expression of a transgene reveals specialized features of cells associated with neuromuscular junctions

After skeletal muscle is denervated, fibroblasts near neuromuscular junctions proliferate more than fibroblasts distant from synaptic sites, and they accumulate adhesive molecules such as tenascin (Gatchalian, C. L., M. Schachner, and J. R. Sanes. 1989. J. Cell Biol. 108:1873-1890). This response could reflect signals that arise perisynaptically after denervation, preexisting differences between perisynaptic and extrasynaptic fibroblasts, or both. Here, we describe a line of transgenic mice in which patterns of transgene expression provide direct evidence for differences between perisynaptic and extrasynaptic fibroblasts in normal muscle. Transgenic mice were generated using regulatory elements from a major histocompatibility complex (MHC) class I gene linked to the Escherichia coli beta-galactosidase (lacZ) gene. Expression of lacZ was detected histochemically. In each of eight lines, lacZ was detected in different subsets of cells, none of which included lymphocytes. In contrast, endogenous MHC is expressed in most tissues and at high levels in lymphocytes. Thus, the MHC gene sequences appeared inactive in the transgene, and lacZ expression was apparently controlled by genomic regulatory elements that were specific for the insertion site. In one line, cells close to the neuromuscular junction were lacZ positive in embryonic and young postnatal mice. Electron microscopy identified these cells as fibroblasts and Schwann cells associated with motor nerve terminals, as well as endoneurial fibroblasts, perineurial cells, and Schwann cells in the distal branches of motor nerves. No intramuscular cells greater than 200 microns from synaptic sites were lacZ positive. These results indicate that there are molecular differences between perisynaptic and extrasynaptic fibroblasts even in normal muscle and that diverse perisynaptic cell types share a specific pattern of gene expression.     

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.     

PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression

Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway.     

Motor neurons contain agrin-like molecules

Molecules antigenically similar to agrin, a protein extracted from the electric organ of Torpedo californica, are highly concentrated in the synaptic basal lamina of neuromuscular junctions in vertebrate skeletal muscle. On the basis of several lines of evidence it has been proposed that agrin-like molecules mediate the nerve-induced formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers at developing and regenerating neuromuscular junctions and that they help maintain these postsynaptic specializations in the adult. Here we show that anti-agrin monoclonal antibodies selectively stain the cell bodies of motor neurons in embryos and adults, and that the stain is concentrated in the Golgi apparatus. We also present evidence that motor neurons in both embryos and adults contain molecules that cause the formation of AChR and AChE aggregates on cultured myotubes and that these AChR/AChE-aggregating molecules are antigenically similar to agrin. These findings are consistent with the hypothesis that agrin-like molecules are synthesized by motor neurons, and are released from their axon terminals to become incorporated into the synaptic basal lamina where they direct the formation of synapses during development and regeneration.     

The single-channel basis for the slow kinetics of synaptic currents in vertebrate slow muscle fibers

The time course of synaptic currents is significantly longer in slow than in fast twitch muscle fibers. To examine the underlying basis for these slow synaptic currents, single-channel recordings were made from the synapses of slow muscle fibers. Our analysis indicates that low conductance acetylcholine receptor (AChR) channels predominate in innervated slow fibers. The high level of expression of low conductance channels is in contrast to fast twitch fibers, in which these channels are expressed in significant numbers only in embryonic or denervated muscle. Analysis of the distribution of open durations for the low conductance channel class suggests that the open time of this AChR class is the major determinant in shaping the slow time course of synaptic current decay. The predominant contribution of low conductance channel openings to synaptic currents of slow muscle fibers indicates a well-defined physiological role for this class of AChRs.     

Essential roles of the acetylcholine receptor gamma-subunit in neuromuscular synaptic patterning

Formation of the vertebrate neuromuscular junction (NMJ) takes place in a stereotypic pattern in which nerves terminate at select sarcolemmal sites often localized to the central region of the muscle fibers. Several lines of evidence indicate that the muscle fibers may initiate postsynaptic differentiation independent of the ingrowing nerves. For example, nascent acetylcholine receptors (AChRs) are pre-patterned at select regions of the muscle during the initial stage of neuromuscular synaptogenesis. It is not clear how these pre-patterned AChR clusters are assembled, and to what extent they contribute to pre- and post-synaptic differentiation during development. Here, we show that genetic deletion of the AChR gamma-subunit gene in mice leads to an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis. The absence of pre-patterned AChR clusters was associated with excessive nerve branching, increased motoneuron survival, as well as aberrant distribution of acetylcholinesterase (AChE) and rapsyn. However, clustering of muscle specific kinase (MuSK) proceeded normally in the gamma-null muscles. AChR clusters emerged at later stages owing to the expression of the AChR epsilon-subunit, but these delayed AChR clusters were broadly distributed and appeared at lower level compared with the wild-type muscles. Interestingly, despite the abnormal pattern, synaptic vesicle proteins were progressively accumulated at individual nerve terminals, and neuromuscular synapses were ultimately established in gamma-null muscles. These results demonstrate that the gamma-subunit is required for the formation of pre-patterned AChR clusters, which in turn play an essential role in determining the subsequent pattern of neuromuscular synaptogenesis.     

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.     

Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α₁subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ?-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ?-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro.     

Shp2 is dispensable in the formation and maintenance of the neuromuscular junction

SHP2, a protein tyrosine phosphatase with two SH2 domains, has been implicated in regulating acetylcholine receptor (AChR) gene expression and cluster formation in cultured muscle cells. To understand the role of SHP2 in neuromuscular junction (NMJ) formation in vivo, we generated mus cle-specific deficient mice by using a loxP/Cre strategy since Shp2 null mutation causes embryonic lethality. Shp2(floxed/floxed) mice were crossed with mice expressing the Cre gene under the control of the human skeletal alpha-actin (HSA) promoter. Expression of SHP2 was reduced or diminished specifically in skeletal muscles of the conditional knockout (CKO) mice. The mutant mice were viable and fertile, without apparent muscle defects. The mRNA of the AChR alpha subunit and AChR clusters in CKO mice were localized in a narrow central region surrounding the phrenic nerve primary branches, without apparent change in intensity. AChR clusters colocalized with markers of synaptic vesicles and Schwann cells, suggesting proper differentiation of presynaptic terminals and Schwann cells. In comparison with age-matched littermates, no apparent difference was observed in the size and length of AChR clusters in CKO mice. Both the frequency and amplitude of mEPPs in CKO mice were similar to those in controls, suggesting normal neurotransmission when SHP2 was deficient. These results suggest that Shp2 is not required for NMJ formation and/or maintenance.     

Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle

Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (alpha2beta(gamma)delta) and adult types of AChR (alpha2beta(epsilon)delta). Mice lacking the epsilon-subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscle differentiation and for the formation of the neuromuscular synapses. Here, we show that substitution of the fetal-type AChR with an adult-type AChR preserves myoblast fusion, muscle and end-plate differentiation, whereas it substantially alters the innervation pattern of muscle by the motor nerve. Mutant mice form functional neuromuscular synapses outside the central, narrow end-plate band region in the diaphragm, with synapses scattered over a wider muscle territory. We suggest that one function of the fetal type of AChR is to ensure an orderly innervation pattern of skeletal muscle.     

Synaptic differentiation is defective in mice lacking acetylcholine receptor beta-subunit tyrosine phosphorylation

Agrin activates MuSK, a receptor tyrosine kinase expressed in skeletal muscle, leading to tyrosine phosphorylation of the acetylcholine receptor (AChR) beta-subunit and clustering of AChRs. The importance of AChR beta-subunit tyrosine phosphorylation in clustering AChRs and regulating synaptic differentiation is poorly understood. We generated mice with targeted mutations in the three intracellular tyrosines of the AChR beta-subunit (AChR-beta(3F/3F)). Mice lacking AChR beta-subunit tyrosine phosphorylation thrive postnatally and have no overt behavioral defects, indicating that AChR beta-subunit tyrosine phosphorylation is not essential for the formation of neuromuscular synapses. Nonetheless, the size of synapses and the density of synaptic AChRs are reduced in AChR- beta(3F/3F) mutant mice. Moreover, synapses are structurally simplified and the organization of postjunctional folds is aberrant in mice lacking tyrosine phosphorylation of the AChR beta-subunit. Furthermore, mutant AChRs cluster poorly in response to agrin and are readily extracted from the cell surface of cultured myotubes by non-ionic detergent. These data indicate that tyrosine phosphorylation of the AChR beta-subunit has an important role in organizing AChRs and regulating synaptic differentiation.