Visualization of Cranial Motor Neurons in Live Transgenic Zebrafish

Visualization of Cranial Motor Neurons in Live Transgenic Zebrafish

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Unformatted text preview: Visualization of Cranial Motor Neurons in Live Transgenic Zebrafish Expressing Green Fluorescent Protein Under the Control of the Islet-1 Promoter/Enhancer Shin-ichi Higashijima, 1,2 Yoshiki Hotta, 3 and Hitoshi Okamoto 4 1 Inheritance and Variation Group, Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Honmachi, Kawaguchi, Saitama 332-0012, Japan, 2 Division of Morphogenesis, National Institute for Basic Biology, Myodaijicho, Okazaki, Aichi 444-8585, Japan, 3 National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan, and 4 Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), Hirosawa, Wako, Saitama 351-0198, Japan We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences to Islet-1 promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and cap- ture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding sug- gests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development. Key words: zebrafish; neuron-specific promoter; transgenic; Islet-1 ; motor neuron; GFP; live visualization Developing motor neurons in the vertebrate hindbrain are an attractive system in which to study mechanisms of segmentation and the processes regulating specific axonal pathfinding (Guthrie, 1996; Lumsden and Krumlauf, 1996; Pfaff and Kintner, 1998). Chick and mouse are the most frequently used experimental model organisms to study motor neuron development in verte- brates. Recently, the zebrafish has emerged as another useful model system. Several features of zebrafish make them especially amenable for developmental studies, including development of the hindbrain. The embryos are transparent, making it possible to readily visualize internal structures and cells in living zebrafish. The embryonic hindbrain is relatively simple and manipulable (Chandrasekhar et al., 1997, 1998), and the zebrafish is amenable to large-scale genetic studies (Driever et al., 1996; Haffter et al., 1996) that have generated interesting mutations in hindbrain motor neurons (Chandrasekhar et al., 1999)....
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This note was uploaded on 05/28/2010 for the course BIOLOGY 03234 taught by Professor Sochacka during the Spring '10 term at Ghent University.

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Visualization of Cranial Motor Neurons in Live Transgenic Zebrafish

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