title: A modified acetylcholine receptor delta-subunit enables a null mutant to survive beyond sexual maturation.
abstract: 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 delta-subunit in zebrafish (sofa potato; sop) leads to the death of embryos around 5 d postfertilization (dpf). In sop(-/-) mutants, we expressed modified delta-subunits, with one (delta1YFP) or two yellow fluorescent protein (delta2YFP) molecules fused at the intracellular loop, under the control of an alpha-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 delta2YFP. These delta2YFP/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.
authors.affiliation: The Whitney Laboratory for Marine Bioscience, University of Florida, St Augustine, Florida 32080, USA.
authors.forename: Kimberly E
authors.initials: KE
authors.lastname: Epley
authors.forename: Jason M
authors.initials: JM
authors.lastname: Urban
authors.forename: Takanori
authors.initials: T
authors.lastname: Ikenaga
authors.forename: Fumihito
authors.initials: F
authors.lastname: Ono
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D005247:Feeding Behavior / Q000502:physiology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D008136:Longevity / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D009154:Mutation / Q000235:genetics*; D009469:Neuromuscular Junction / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D020511:Neuromuscular Junction Diseases / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011950:Receptors, Cholinergic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012726:Sexual Behavior, Animal / Q000502:physiology; D012741:Sexual Maturation / Q000235:genetics; D013570:Synaptic Membranes / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D021122:Protein Subunits; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D000109:Acetylcholine