N ATU RE N EU RO SCI EN CE VOLUME 7 | NUMBER 12 | DECEMBER 2004 1303 ARTICLES The assembly of neural connections depends on the precision with which growth cones navigate to their synaptic partners. En route to their tar- gets, growth cones encounter a series of choice points, at which indi- vidual growth cones choose a path specific for their neuronal subtype. Specialized cells at the central nervous system midline provide a well- characterized repertoire of signals that control divergent axonal behav- iors 1,2 . In contrast, much less is known about the cell types and molecules that direct individual motor axons at peripheral choice points. It is clear that trajectory choice by motor axons is dictated by neuronal expression of cell-specific transcription factors, but also critically depends on extrin- sic cues 3,4 . For example, the plexus region at the base of the avian limb serves as an intermediate target at which individual motor neuron popu- lations make divergent pathway choices 5 . Elegant microsurgical manipu- lations in the chick have suggested the existence of guidance cues in this area 6,7 . However, with a few exceptions such as mesenchymal ephrin A directing EphA-positive motor axons 8,9 , little is known about extrinsic signals that control vertebrate motor axon guidance. Studies on motor pathway choice in the zebrafish have the advantage of single-cell resolution. Each of the three segmental primary motor neurons projecting into axial muscle has a distinct spinal cord position, expresses a unique combination of LIM homeodomain transcription factors and has a stereotyped axonal trajectory 10 . Initially, all three motor growth cones extend along the medial surface of the somites toward a somitic choice point 11,12 . Here, all growth cones first pause and make extensive contacts with specialized cells called muscle pioneers 13,14 , then select a cell-type specific path to ventral, dorsal or medial myotomal regions ( Fig. 1a). We have previously shown that the unplugged gene, through a non–cell- autonomous mechanism, provides competence for motor growth cones to navigate this choice point 15 . In unplugged-null mutants, two of the three motor neurons fail to select their cell type–specific path. Chimera studies showed that unplugged is dispensable in muscle pioneer cells located at the choice point but is required in dorsal adaxial cells 15 . Initially, these dorsal adaxial cells delineate the axonal path leading to the choice point, but as motor growth cones approach, they migrate away 16,17 (Fig. 1). Based on these results, we proposed that, before their migration, unplugged enables dorsal adaxial cells to modify the extracellular matrix (ECM) adjacent to the choice point, thereby providing competence to growth cones to select the correct path before reaching the upcoming choice point 15 (Fig. 1). Here, we report the positional cloning of the unplugged gene and show that it encodes a receptor tyrosine kinase most similar to MuSK and their related orphan receptors (RORs) 18 . In mammals, MuSK is essential for the assembly of neuromuscular junctions, where it responds to agrin signals to recruit the intracellular linker rapsyn and cluster acetylcho- line receptors (AChRs) 19,20 . We show in the zebrafish that functional neuromuscular synapses develop in complete absence of the unplugged gene. We find that alternative splicing generates three different unplugged transcripts, and that one of these isoforms lacks the extracellular modules mediating agrin-induced AChR clustering. We demonstrate that signal- ing through this unplugged isoform occurs independently of rapsyn and is essential for motor axons to choose their future path. Our results uncover a specific and previously unknown role of MuSK homologs in motor axonal pathway selection. RESULTS Positional cloning of unplugged We had previously mapped the unplugged locus to a small interval 1 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058, USA. 2 Present address: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. 3 These authors contributed equally to this work. Correspondence should be addressed to M.G. (granatom@mail.med.upenn.edu). Published online 14 November 2004; doi:10.1038/nn1350 Zebrafish unplugged reveals a role for muscle- specific kinase homologs in axonal pathway choice Jing Zhang 1–3 , Julie L Lefebvre 1,3 , Shuxia Zhao 1,3 & Michael Granato 1 En route to their target, pioneering motor growth cones repeatedly encounter choice points at which they make pathway decisions. In the zebrafish mutant unplugged, two of the three segmental motor axons make incorrect decisions at a somitic choice point. Using positional cloning, we show here that unplugged encodes a homolog of muscle-specific kinase (MuSK) and that, unlike mammalian MuSK, unplugged has only a limited role in neuromuscular synaptogenesis. We demonstrate that unplugged is transiently expressed in cells adjacent to the choice point and that unplugged signaling before the arrival of growth cones induces changes in the extracellular environment. In addition, we find that the unplugged locus generates three different transcripts. The splice variant 1 (SV1) isoform lacks the extracellular modules essential for agrin responsiveness, and signaling through this isoform mediates axonal pathfinding, independent of the MuSK downstream component rapsyn. Our results demonstrate a new role for MuSK homologs in axonal pathway selection. © 2004 Nature Publishing Group http://www.nature.com/natureneuroscience