In FY2009, our section continued projects focused on the nervous system. All experiments use the nervous system of zebrafish as the experimental model. Some experiments focus on the neuromuscular junction, and others directly examine the brain. With regard to the alcohol research, we started to examine the change of nervous system in ethanol-treated zebrafish embryos in collaboration with Dr. Harold Burgess at NICHD. All projects combine genetics, confocal microscopy, molecular biology and cellular physiology. Through most of FY09, the section had four people including the PI. Dr.Takanori Ikenaga, who worked as a visiting fellow, obtained the position of assistant professor in the University of Hyogo, Japan, and left in 03/2009. A new visiting fellow, Dr. Jee-Young Park, joined the lab in 05/2009. We presented three posters in the Neuroscience Meeting held at Washington DC, one poster in the ARVO meeting held in Fort Lauderdale, FL, a poster and two talks in the 6th European Zebrafish meeting held in Rome, Italy, and a poster in the NIH research festival. These presentations include results of collaborations with Dr. Paul Brehm at the Oregon Health and Science University, Dr.Alan Davidson at Harvard University, Dr.Koichi Kawakami at the National institute of genetics in Japan, Dr.Ralph Nelson at NINDS, and Dr. Victoria Connaughton at the American University. In 12/2008, we published a paper in the Journal of Neuroscience entitled: A modified acetylcholine receptor subunit enables a null mutant to survive beyond the sexual maturation. The abstract is as follows: The contraction of skeletal muscle is dependent upon the 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 with 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 days post-fertilization (dpf). In sop-/- mutants, we expressed modified delta-subunits, with one (1YFP) or two yellow fluorescent protein (2YFP) molecules fused at the intracellular loop. 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 stably-rescued line of sop-/-. These 2YFP/sop-/- embryos can mount escape behavior close to wild type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared to wild type fish. Remarkably, these embryos grow to adulthood and display normal locomotion, including such complex behaviors 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. Altered synaptic strength resulting from the transgene delineates requirements for gene therapies of NMJ. Another manuscript currently under review is entitled: Formation of spinal network in zebrafish determined by domain-specific Pax genes. The abstract is as follows: In the formation of the spinal network, various transcription factors interact to develop specific cell types. Using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) trapped the pax8 gene. RFP insertion marked pax8-lineage cells and abolished pax8 expression in homozygous embryos. Pax8 and pax2a are expressed in dorsal regions of the spinal cord. We examined the effect of pax genes on interneuron development. In pax8 mutants, the pax8-lineage cells undergo normal differentiation and the swimming behavior remained intact. In contrast, in pax2a/pax8 double mutants, the pax8-lineage cells displayed altered cell fates. Circumferential Descending (CiD) interneurons, which never express pax8 in normal embryos, arose from the pax8-lineage. The use of newly identified gene trap line revealed roles of transcription factors in the network formation of the spinal cord.
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