Overexpression of muscle specific kinase increases the transcription and aggregation of acetylcholine receptors in Xenopus embryos

Abstract

Muscle specific kinase (MuSK) mediates agrin-induced acetylcholine receptor (AChR) aggregation on muscle membrane at the neuromuscular junction (NMJ). To examine whether MuSK enhances NMJ formation during embryonic development in vivo, the level of expression of MuSK was manipulated in Xenopus embryos and the functional consequence at the NMJ was assessed. We found that overexpression of MuSK enhanced the formation of NMJ by increasing the aggregation of AChRs at innervated regions in developing embryos. The area of AChR aggregation increased by ∼2-fold in MuSK injected embryos during the critical stages of NMJ formation. Interestingly, overexpression of MuSK in Xenopus embryos was found to induce the level of AChR transcript. Deletion of the Kringle domain in the MuSK construct did not attenuate the observed induction of AChR transcription and aggregation. Taken together, our findings provide the first demonstration that increased level of MuSK expression in vivo significantly elevate the aggregation and transcription of AChR at the NMJ in developing Xenopus embryos.

Introduction

Reciprocal signaling between motor neurons and muscle fibers is critical for the formation of the neuromuscular junction (NMJ; [23]). One of the hallmarks for postsynaptic specializations during the formation of NMJ is the clustering of acetylcholine receptors (AChRs) on muscle fibers. The mechanisms controlling the aggregation of AChRs are at both transcriptional and post-translational levels. While neuregulin is known to induce transcription of specific genes at subsynaptic nuclei on muscle, agrin is well documented to exert the post-translational control by inducing the clustering of pre-existing AChRs that are initially distributed throughout the muscle membrane [4].

The activation of a muscle-specific receptor tyrosine kinase, MuSK, represents a critical step in the signaling pathway of agrin. The expression of MuSK is precisely colocalized with AChRs at the synapse of adult skeletal muscle [7], [25] and MuSK has been shown to mediate agrin-induced tyrosine phosphorylation and aggregation of AChRs in cultured myotubes [12]. Furthermore, MuSK knockout mice die shortly after birth and do not exhibit the aggregation of AChRs at the NMJ [6], a phenotype similar to that observed in agrin knockout mice [10].

The Xenopus system is used in the present study to elucidate the functional roles of MuSK in the formation of NMJ during embryonic development. Sequence analysis of Xenopus MuSK identified in our laboratory reveals similarity with all known forms of MuSK, including Torpedo, human, mouse, rat and chicken [7], [9], [16], [17], [25]. Interestingly, the cloning of MuSK from Torpedo, Xenopus and chicken demonstrates shared characteristic features that are different from the known mammalian counterparts. For example, just like chicken and Torpedo MuSK, Xenopus MuSK contains a Kringle domain between the Ig-like IV domain and the transmembrane domain. Moreover, the expression of Xenopus MuSK is not restricted to muscle but can also be detected in adult spleen and developing central nervous system, including brain, eye and spinal cord. Similarly, chicken MuSK can be detected in the cerebellum during early embryonic development.

The well-characterized rapid development of NMJ and the external embryonic development in Xenopus provide a useful in vivo system to study the effects of ectopically expressed synaptic proteins during the formation of NMJ. In this study, the functional consequences of overexpressing wild type MuSK and its Kringle-domain deleted (K-del) mutant were examined at the innervated regions in the injected embryos. We report here that an induction of the AChR transcription and aggregation was observed at the NMJ in Xenopus embryos injected with wild type or K-del MuSK mutant, suggesting that MuSK enhanced the formation of NMJ during Xenopus embryonic development.