In 2014, three papers were published from the lab. A paper by Fero et al., a collaboration with Dr. Burgess at NICHD, was published in Disease Models and Mechanisms. In this paper, we identified an early embryonic motility mutant in zebrafish caused by integration of a transgene into the pseudophosphatase dual specificity phosphatase 27 (dusp27) gene. Dusp27 mutants exhibit near complete paralysis at embryonic and larval stages, producing extremely low levels of spontaneous coiling movements and a greatly diminished touch response. Loss of dusp27 does not prevent somitogenesis but results in severe disorganization of the contractile apparatus in muscle fibers. Sarcomeric structures in mutants are almost entirely absent and only rare triads are observed. These findings are the first to implicate a functional role of dusp27 as a gene required for myofiber maturation and provide an animal model for analyzing the mechanisms governing myofibril assembly. Another paper by Won et al. , in collaboration with Dr. Ikeda at NIAAA, was published in PLoS ONE. In this paper, RGK proteins were studied. RGK proteins are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel βsubunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein-Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein--Cavβsubunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggested that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago. A third paper by Park et al. was published in the Journal of Neuroscience. We reported in this paper a zebrafish mutant of the AChR δsubunit that exhibits two distinct NMJ phenotypes specific to two muscle fiber types: slow or fast. Mutations in AChR subunits, expressed as pentamers in neuromuscular junctions (NMJs), cause various types of congenital myasthenic syndromes. In AChR pentamers, the adult εsubunit gradually replaces the embryonic γsubunit as the animal develops. Because of this switch in subunit composition, mutations in specific subunits result in synaptic phenotypes that change with developmental age. However, a mutation in any AChR subunit is considered to affect the NMJs of all muscle fibers equally. Homozygous fish harboring a newly identified point mutation in the δsubunit formed functional AChRs in slow muscles, whereas receptors in fast muscles were nonfunctional. To test the hypothesis that different subunit compositions in slow and fast muscles underlie distinct phenotypes, we examined the presence of ε/γsubunits in NMJs using specific antibodies. Both wild-type and mutant larvae lacked ε/γsubunits in slow muscle synapses. These findings in zebrafish suggested that some mutations in human congenital myasthenic syndromes may affect slow and fast muscle fibers differently. The lab will move to the Osaka Medical College in 2015, and in 2014 we prepared for the transfer. Several projects, some mainly performed in the lab and others in collaboration with multiple labs at NIH, will be completed soon or will be continued in a new lab at Osaka. Personnel in the lab presented on-going works in several meetings, including the Mid-Atlantic Regional Zebrafish Meeting and the 11th International Conference on Zebrafish Development and Genetics. Fumihito Ono gave invited talks at the University of Osaka, National Eye Institute and the NIH research festival symposium.
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