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Generation of dispersed presomitic mesoderm cell cultures for imaging of the zebrafish segmentation clock in single cells.

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Webb,  Alexis
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

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Soroldoni,  Daniele
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

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Oswald,  Annelie
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

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Schindelin,  Johannes
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

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Oates,  Andrew C.
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

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Citation

Webb, A., Soroldoni, D., Oswald, A., Schindelin, J., & Oates, A. C. (2014). Generation of dispersed presomitic mesoderm cell cultures for imaging of the zebrafish segmentation clock in single cells. Journal of Visualized Experiments: Jove, (89): e50307.


Cite as: https://hdl.handle.net/21.11116/0000-0001-0584-4
Abstract
Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.