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 Ϫ/Ϫ 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 Ϫ/Ϫ fish stably expressing the ␦2YFP. These ␦2YFP/sop Ϫ/Ϫ 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 IntroductionThe synapse between the motor nerve and skeletal muscle, commonly referred to as the neuromuscular junction (NMJ), is cholinergic in vertebrates and is a model system for the investigation of synapses (Sanes and Lichtman, 2001;Ono, 2008). On the postsynaptic face of the NMJ, the AChRs span the membrane, with each AChR forming a pentameric structure. The five subunits comprising AChRs are 2␣s, , ␦ and ␥/. ␥ and are developmentally regulated and can substitute for each other. The embryonic-type ␥ is replaced by the adult-type as the synapse matures (Mishina et al., 1986).A group of genetic disorders have mutations in genes coding for components of the NMJ. Congenital myasthenic disorders (CMDs) result from mutations in genes coding for expression of proteins such as AChR, rapsyn, MuSK, and cholinesterase (Engel and Sine, 2005). CMD patients display weak muscle strength resulting from compromised synaptic currents. Functional nulls of AChRs were predicted to be lethal and, indeed, fetuses homozygous for these mutations die in the first trimester (Michalk et al., 2008). Affected fetuses display characteristic anatomical features that are collectively called Fetal Akinesia Deformation Sequences (FADS).Zebrafish (Danio rerio)...
The formation and function of synapses are tightly orchestrated by the precise timing of expression of specific molecules during development. In this study, we determined how manipulating the timing of expression of postsynaptic acetylcholine receptors (AChRs) impacts presynaptic release by establishing a genetically engineered zebrafish line in which we can freely control the timing of AChR expression in an AChR-less fish background. With the delayed induction of AChR expression after an extensive period of AChR-less development, paralyzed fish displayed a remarkable level of recovery, exhibiting a robust escape response following developmental delay. Despite their apparent behavioral rescue, synapse formation in these fish was significantly altered as a result of delayed AChR expression. Motor neuron innervation determined the sites for AChR clustering, a complete reversal of normal neuromuscular junction (NMJ) development where AChR clustering precedes innervation. Most importantly, among the three modes of presynaptic vesicle release, only the spontaneous release machinery was strongly suppressed in these fish, while evoked vesicle release remained relatively unaffected. Such a specific presynaptic change, which may constitute a part of the compensatory mechanism in response to the absence of postsynaptic AChRs, may underlie symptoms of neuromuscular diseases characterized by reduced AChRs, such as myasthenia gravis.The vertebrate NMJ is a cholinergic synapse formed between a motor nerve and skeletal muscle. Zebrafish provide an exceptional model system to observe this synapse formation in vivo because of their transparency, rapid development, and the various genetic tools available to alter their synaptic function. In zebrafish embryos, neuromuscular synapses start to function and lead to spontaneous locomotion around 17 hours post fertilization (hpf) 1 . We previously analyzed a mutant zebrafish that lacked AChRs in the NMJ and found the morphology of its presynaptic terminals was largely normal 2 . However, our analysis of the functional consequences of losing postsynaptic AChRs on the presynaptic machinery was limited because synaptic currents were not measurable 3 .In order to address this issue, here we have established a genetically engineered zebrafish line in which we can freely control the timing of AChR expression in an AChR-less fish background using a chemically inducible gene expression system. By allowing a sufficient time lag before inducing the expression of AChRs, we could observe the effect of AChR-less development on NMJ synaptic currents. We found that these synapses exhibited remarkable adaptability, which inevitably led to functional transmission. However, these rescued synapses manifested characteristics that were remarkably distinct from their normally developed counterparts, namely the lack of spontaneous vesicle releases. These differences impacted their swimming performance.
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