期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 89, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/50307
关键词
Developmental Biology; Issue 89; Zebrafish; Primary Cell Culture; Biological Clocks; Somitogenesis; Oscillator; In Vitro; Time-lapse Imaging; Primary Culture; Fluorescence
资金
- EMBO Long-term fellowship
- National Science Foundation International Postdoctoral Research Fellowship
- Max Planck Gesellschaft
- DIGS-BB fellowship
- European Research Council under the European Communities Seventh Framework Programme [STG-207634]
- Medical Research Council [MC_UP_1202/3] Funding Source: researchfish
- MRC [MC_UP_1202/3] Funding Source: UKRI
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.
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