Neuromuscular synapse formation in mice lacking motor neuron- and skeletal muscle-derived Neuregulin-1

Abstract

The localization of acetylcholine receptors (AChRs) to the vertebrate neuromuscular junction is mediated, in part, through selective transcription of AChR subunit genes in myofiber subsynaptic nuclei. Agrin and the muscle-specific receptor tyrosine kinase, MuSK, have critical roles in synapse-specific transcription, because AChR genes are expressed uniformly in mice lacking either agrin or MuSK. Several lines of evidence suggest that agrin and MuSK stimulate synapse-specific transcription indirectly by regulating the distribution of other cell surface ligands, which stimulate a pathway for synapse-specific gene expression. This putative secondary signal for directing AChR gene expression to synapses is not known, but Neuregulin-1 (Nrg-1), primarily based on its presence at synapses and its ability to induce AChR gene expression in vitro, has been considered a good candidate. To study the role of Nrg-1 at neuromuscular synapses, we inactivated nrg-1 in motor neurons, skeletal muscle, or both cell types, using mice that express Cre recombinase selectively in developing motor neurons or in developing skeletal myofibers. We find that AChRs are clustered at synapses and that synapse-specific transcription is normal in mice lacking Nrg-1 in motor neurons, myofibers, or both cell types. These data indicate that Nrg-1 is dispensable for clustering AChRs and activating AChR genes in subsynaptic nuclei during development and suggest that these aspects of postsynaptic differentiation are dependent on Agrin/MuSK signaling without a requirement for a secondary signal.

Figure 1.

Neuronal Nrg-1 is dispensable for normal clustering of AChRs at synapses. Whole mounts of diaphragm muscles from E18.5 neuronal nrg-1 mutant embryos (B) and control littermates (A) were stained with Alexa594-α-bungaratoxin to visualize postsynaptic AChRs (red), antibodies against neurofilament and synaptophysin to visualize motor axons and nerve terminals (green), respectively, and Alexa660-phalloidin to visualize muscle fibers (blue). A, In control mice, postsynaptic AChR clusters are apposed to nerve terminals. B, In mice lacking neuronal Nrg-1, motor axons have withdrawn, but AChRs remain clustered at synaptic sites, and AChR clusters are of similar size and shape as AChR clusters in control mice. Scale bar: (in B) A, B, 5 μm. C, Synaptic AChR density and total synaptic AChR levels are indistinguishable in neuronal nrg-1 mutant mice (n = 4) and control littermates (n = 5). Error bars indicate SEM.

Figure 2.

Efficient Cre-mediated deletion of loxP-flanked target genes in skeletal muscle of HSA::cre mice. Cross sections of gastrocnemius, diaphragm, and intercostal muscles from Z/AP and HSA::cre; Z/AP mice were stained to visualize and measureβ-gal activity. The reaction was terminated at different time points, and grayscale values from muscle fibers were analyzed to measure product accumulation and determine β-gal activity (see Materials and Methods). A, B, Representative images of intercostal muscles stained for 1 h. C, After a 10 h reaction, interstitial cells, located between myofibers, are intensely stained (arrow), and diffusion of this stain is likely to contribute to product accumulation in adjacent myofibers. Scalebar: (inC) A, B, 10 μm; C, 8 μm. D, Plotted grayscale values (with SDs) for intercostal muscles from a single Z/AP and a single HSA::cre; Z/AP mouse. E, β-gal activity in muscle of two Z/AP mice (the average of the 2 mice was assigned a value of 100%) and two HSA::cre; Z/AP mice. The HSA::cre transgene reduces β-gal activity (>95%) in all muscle groups.

Figure 3.

Muscle-derived Nrg-1 is not required for motor innervation and clustering AChRs at synapses. Whole mounts of diaphragm muscles from E18.5 embryos lacking muscle-derived Nrg-1 (B, D) and control littermates (A, C) were stained as described in Figure 1. A-D, In control mice and mice lacking muscle-derived Nrg-1, the main intramuscular nerve is positioned in the central region of the diaphragm muscle (arrow in A), and individual motor axon branches innervate muscle fibers adjacent to the main nerve trunk. C, D, Nerve terminals are apposed to AChR clusters of normal appearance. Scale bar: (in D) A, B, 125 μm; C, D, 5 μm. E, Synaptic AChR density and total synaptic AChR levels in nrg-1 mutant mice (n = 5) and control littermates (n = 5) are not significantly different. Error bars indicate SEM.

Figure 4.

Synapses mature normally in the absence of muscle-derived Nrg-1. A, B, Whole mounts of diaphragm muscles from P21 control mice and mice lacking muscle-derived Nrg-1 were stained as described in Figure 1. By P21, the shape of the neuromuscular synapse has matured from a plaque-like to a pretzel-like structure in control mice and mice lacking muscle-derived Nrg-1. C, The density and total number of synaptic AChRs is similar in muscle from control mice (n=3) and mice lacking muscle-derived Nrg-1 (n=4). Error bars indicate SEM. D, E, Whole mounts of intercostal muscles from P21 control mice (D) and mice lacking muscle-derived Nrg-1 (E) were processed for in situ hybridization. AChR ϵ mRNA is concentrated in the central region of muscle from nrg-1 mutant P21 mice, similar to control mice. M indicates the position of the muscle, and R indicates the location of the ribs. Scale bar: (in E) A, B, 10 μm; D, E, 200 μm.

Figure 5.

Synapse-specific gene expression is normal in mice lacking Nrg-1 in motor neurons and skeletal muscle. A-H, Whole mounts of intercostal muscles from E18.5 embryos lacking neuronal- and muscle-derived Nrg-1 (A, C, E, G) and control (B, D, F, H) mice were processed for in situ hybridization. AChRα subunit (A, B), AChRδ subunit (C, D), MuSK (E, F), and Utrn (G, H) mRNAs are concentrated in the central region of muscle from mice lacking Nrg-1 in motor neurons and skeletal muscle, like in control mice. Scale bar (in H) A-H, 200 μm. I, RNA from gastrocnemius muscles of E18.5 embryos lacking neuronal and muscle-derived Nrg-1 and from control mice was reverse transcribed into cDNA, and gene expression were measured by real-time PCR. The expression levels of AChR δ and MuSK are similar in mice lacking Nrg-1 in motor neurons and muscle (n = 3) and in control mice (n = 2). Error bars indicate SEM.

Figure 6.

Synaptic AChR levels are reduced in mice lacking both neuronal- and muscle-derived Nrg-1. Whole mounts of diaphragm muscles from E18.5 embryos lacking neuronal and muscle-derived Nrg-1 (B) and control embryos (A) were stained as described in Figure 1. A, In control mice, AChRs are apposed to nerve terminals. B, In mice lacking neuronal- and muscle-derived Nrg-1, motor axons have withdrawn, but AChRs remain clustered at synaptic sites. A, B, AChR clusters appear morphologically similar in control mice and mice lacking Nrg-1 in neurons and muscle. Scale bar (in B) A, B, 5 μm. C, The density and number of synaptic AChRs is reduced in mice lacking neuronal- and muscle-derived Nrg-1 (n = 8 for doubly, conditional mutant mice; n = 7 for control mice). Error bars indicate SEM. ^*p < 0.02 and ^**p < 0.01 indicate that the values are significantly different from control.