INTRODUCTION The development of striated muscle is dependent on the exchange of signals between the nerve terminals of sensory and motoneurons with developing muscle fibers. Motoneuron derived signals regulate the development of neuromuscular junctions (NMJs) (Burden, 1998; Sanes and Lichtman, 1999). Sensory neuron derived signals are essential for the development of muscle spindles (Maier, 1997; Walro and Kucera, 1999). During formation of the NMJ, synaptic proteins such as acetylcholine receptors (AChRs), rapsyn, Musk, Nrg1 and its Erbb receptors become clustered in the postsynaptic muscle membrane. Furthermore, the expression of genes that encode postsynaptic proteins becomes restricted to subsynaptic nuclei (Burden, 1998; Sanes and Lichtman, 1999). Clustering of proteins and synaptic gene expression are dependent on agrin that is released from motor nerve terminals and activates the Musk receptor tyrosine kinase in muscle. Accordingly, in mice that lack agrin or Musk, the formation of stable synaptic AChR clusters and synaptic gene expression are defective (DeChiara et al., 1996; Gautam et al., 1996). Agrin/Musk is not only necessary, but also sufficient for postsynaptic differentiation. Ectopic expression of agrin or of constitutively active Musk in muscle induces the formation of a postsynaptic apparatus and the upregulation of genes normally restricted to subsynaptic nuclei (Cohen et al., 1997; Jones et al., 1997; Jones et al., 1999; Meier et al., 1997; Moore et al., 2001). Nrg1 and its Erbb receptors have also been implicated in the regulation of gene expression in subsynaptic nuclei. Several Nrg1 isoforms are generated from the Nrg1 gene by alternative splicing. Isoforms containing an EGF domain induce AChR gene expression in cultured myotubes in vitro (Buonanno and Fischbach, 2001; Schaeffer et al., 2001). Mice heterozygous for a targeted mutation ablating an Ig-domain encoding exon of the Nrg1 gene have decreased numbers of synaptic AChRs, suggesting that Ig-domain containing Nrg1 isoforms are important in vivo (Sandrock et al., 1997). The cellular source of Nrg1 that regulates synaptic gene expression is at present unclear. Mice that lack Nrg1 specifically in motoneurons still develop NMJs (Schaeffer et al., 2001; Yang et al., 2001), suggesting that Nrg1 may be provided by another cellular source, e.g. by muscle fibers. Consistent with this interpretation, Nrg1/Erbb receptors are clustered at postsynaptic sites induced by ectopic expression of agrin in muscle fibers in the absence of nerve terminals (Jones et al., 1996; Jones et al., 1999; Meier et al., 1998; Moore et al., 2001). Interestingly, expression of synapse specific genes in ectopically induced postsynaptic membranes is decreased when the function of Erbb2 or Erbb4 is blocked in muscle (Jones et al., 1996; Jones et al., 1999; Meier et al., 1998; Moore et al., 2001). Nrg1/Erbb are also concentrated at endogenous NMJs, but fail to cluster in mice that lack agrin or Musk (DeChiara et al., 1996; Gautam et al., 1996). Taken together, these data suggest that muscle derived Nrg1 acts downstream of nerve-derived agrin. 2291 Development 130, 2291-2301 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00447 Neuregulins and their Erbb receptors have been implicated in neuromuscular synapse formation by regulating gene expression in subsynaptic nuclei. To analyze the function of Erbb2 in this process, we have inactivated the Erbb2 gene in developing muscle fibers by Cre/Lox-mediated gene ablation. Neuromuscular synapses form in the mutant mice, but the synapses are less efficient and contain reduced levels of acetylcholine receptors. Surprisingly, the mutant mice also show proprioceptive defects caused by abnormal muscle spindle development. Sensory Ia afferent neurons establish initial contact with Erbb2-deficient myotubes. However, functional spindles never develop. Taken together, our data suggest that Erbb2 signaling regulates the formation of both neuromuscular synapses and muscle spindles. Key words: Erbb2, Nrg1, Muscle spindle, Synapse, Neuromuscular junction, Mouse SUMMARY Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development Marco Leu 1 , Elena Bellmunt 2 , Martin Schwander 1 , Isabel Fariñas 2 , Hans Rudolf Brenner 3 and Ulrich Müller 1, * ,† 1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstr. 66, 4058 Basel, Switzerland 2 Departamento de Biologia Celular, Universidad de Valencia, 46100 Burjassot, Spain 3 Department of Physiology, University of Basel, 4051 Switzerland *Present address: The Scripps Research Institute, 10550 North Torrey Pines Road, ICND 222, La Jolla, CA 92037, USA † Author for correspondence (e-mail: umueller@scripps.edu) Accepted 3 February 2003