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The Journal of Neuroscience, December 3, 2008, 28(49):13223-13231; doi:10.1523/JNEUROSCI.2814-08.2008

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Development/Plasticity/Repair
A Modified Acetylcholine Receptor -Subunit Enables a Null Mutant to Survive Beyond Sexual Maturation

Kimberly E. Epley,^1 * Jason M. Urban,^2 * Takanori Ikenaga,² and Fumihito Ono²

¹The Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, Florida 32080, and ²Section on Model Synaptic Systems, Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism–National Institutes of Health, Bethesda, Maryland 20892

Correspondence should be addressed to Fumihito Ono, MSC9411, National Institute on Alcohol Abuse and Alcoholism–National Institutes of Health–Bethesda, MD 20892. Email: onof{at}mail.nih.gov

The contraction of skeletal muscle is dependent on synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the -subunit in zebrafish (sofa potato; sop) leads to the death of embryos around 5 d postfertilization (dpf). In sop^–/– mutants, we expressed modified -subunits, with one (1YFP) or two yellow fluorescent protein (2YFP) molecules fused at the intracellular loop, under the control of an -actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop^–/– fish stably expressing the 2YFP. These 2YFP/sop^–/– embryos can mount escape behavior close to that of their wild-type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared with wild-type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ.

Key words: zebrafish; neuromuscular junction; acetylcholine receptor; synapse; fetal akinesia deformation sequence; fluorescent protein

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Received June 19, 2008; revised Oct. 14, 2008; accepted Oct. 16, 2008.

Correspondence should be addressed to Fumihito Ono, MSC9411, National Institute on Alcohol Abuse and Alcoholism–National Institutes of Health–Bethesda, MD 20892. Email: onof{at}mail.nih.gov

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