PUBLICATION
Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells
- Authors
- Webb, A.B., Soroldoni, D., Oswald, A., Schindelin, J., Oates, A.C.
- ID
- ZDB-PUB-140801-11
- Date
- 2014
- Source
- Journal of visualized experiments : JoVE (89): (Journal)
- Registered Authors
- Oates, Andrew
- Keywords
- none
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- Biological Clocks/physiology*
- Cell Culture Techniques/methods*
- Female
- Fluorescent Dyes/chemistry
- Male
- Mesoderm/cytology*
- Optical Imaging/methods
- Somites/cytology*
- Time-Lapse Imaging/methods
- Zebrafish/embryology*
- Zebrafish/genetics
- PubMed
- 25078855 Full text @ J. Vis. Exp.
Citation
Webb, A.B., 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).
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping