title: Generation of dispersed presomitic mesoderm cell cultures for imaging of the zebrafish segmentation clock in single cells.
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.
authors: Max Planck Institute of Molecular Cell Biology and Genetics; webb@mpi-cbg.de.
authors: Alexis B
authors: AB
authors: Webb
authors: Max Planck Institute of Molecular Cell Biology and Genetics.
authors: Daniele
authors: D
authors: Soroldoni
authors: Max Planck Institute of Molecular Cell Biology and Genetics.
authors: Annelie
authors: A
authors: Oswald
authors: Max Planck Institute of Molecular Cell Biology and Genetics.
authors: Johannes
authors: J
authors: Schindelin
authors: Max Planck Institute of Molecular Cell Biology and Genetics.
authors: Andrew C
authors: AC
authors: Oates
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001683:Biological Clocks / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D008297:Male; D008648:Mesoderm / Q000166:cytology*; D061848:Optical Imaging / Q000379:methods; D019170:Somites / Q000166:cytology*; D059008:Time-Lapse Imaging / Q000379:methods; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes