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Zebrafish unplugged reveals a role for muscle-specific kinase homologs in axonal pathway choice

Nature Neuroscience volume 7, pages 1303–1309 (2004)Cite this article

Abstract

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.

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Figure 1: Phenotype and model of unplugged.
Figure 2: Cloning and sequence analysis of unplugged.
Figure 3: Neuromuscular synapse formation in wild-type, unplugged and twitch once (rapsyn) mutants.
Figure 4: Morpholino-mediated knockdown of individual unplugged transcripts.
Figure 5: Expression patterns of unplugged full-length and SV1 mRNA.
Figure 6: The unplugged mRNA and protein are concentrated at mature neuromuscular synapses.
Figure 7: Immunostaining for CSPs.

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Acknowledgements

We thank A. Coulson and the Sanger Center for sequencing BAC and PAC clones; P. Brehm for twitch once mutants; and M. Mullins, G. Bashaw, V. Schneider, G. Downes, H. Burgess, M. Gyda and L. Jing for critical comments on the manuscript. This work was supported by a Fellowship from the Myasthenia Gravis Foundation to J.L.L. and grants from the National Institutes of Health to M.G.

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Author notes

  1. Jing Zhang

    Present address: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

  2. Jing Zhang, Julie L Lefebvre and Shuxia Zhao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, 19104-6058, Pennsylvania, USA

    Jing Zhang, Julie L Lefebvre, Shuxia Zhao & Michael Granato

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Corresponding author

Correspondence to Michael Granato.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Sequence alignment between unplugged and MuSK family members. (PDF 77 kb)

Supplementary Fig. 2

unplugged dependent tyrosine phosphorylation on adaxial cells. Confocal images of cross section from 20 somite stage wild-type (a-c, g-i) and unplugged embryos (d-f, j-l) stained with α-pTyr and with the adaxial specific F59 antibody. (a-f) In somites 18-20, phosphotyrosine staining is detectable through out the embryo, but appears concentrated on premigratory adaxial cells. Note the intense phosphotyrosine staining on membranes between adaxial cells (arrowheads in inset) (g-l) In somites 16/15, adaxial cells are about to migrate. In unplugged mutants, phosphotyrosine staining on membranes between adaxial cells is severely reduced (k, l; 15 sections from 5 genotyped -/- embryos). No reduction is detectable on lateral membranes shared with notochord (medial) or fast muscle (lateral), but there seems to be increased phosphotyrosine staining on fast muscle (lateral). (JPG 183 kb)

Supplementary Video 1

Wild-type larvae at 5 dpf. (MOV 207 kb)

Supplementary Video 2

unplugged larvae at 5 dpf. (MOV 158 kb)

Supplementary Methods (PDF 71 kb)

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Zhang, J., Lefebvre, J., Zhao, S. et al. Zebrafish unplugged reveals a role for muscle-specific kinase homologs in axonal pathway choice. Nat Neurosci 7, 1303–1309 (2004). https://doi.org/10.1038/nn1350

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