Home › Cell Biology › Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells
Cell Biology JoVE (Open Access) Citable · DOI

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

DOI: 10.3791/50307-v
What you'll learn
  • ✓ Dissect and disperse zebrafish presomitic mesoderm tissue into single-cell suspensions
  • ✓ Image oscillating fluorescent segmentation clock reporters in live cells over time
  • ✓ Process time-lapse microscopy data to quantify cyclic gene expression patterns
  • ✓ Interpret single-cell dynamics of the zebrafish segmentation clock in vitro
Protocol

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Difficulty
advanced
Total time
~3–4 hours per embryo dissection and imaging session
Model organism
Zebrafish (Danio rerio) transgenic lines
Biosafety
BSL-1

Steps

1
Prepare dissection tools and embryo staging

Sterilize and arrange dissection instruments, stage transgenic zebrafish embryos at the presomitic mesoderm developmental stage, and prepare imaging media.

▶ 01:39
2
Isolate presomitic mesoderm tissue from embryos

Dissect zebrafish embryos under a stereomicroscope and carefully extract the presomitic mesoderm region using fine needles and surgical techniques.

▶ 03:15
3
Disperse tissue into single cells

Mechanically and enzymatically dissociate presomitic mesoderm tissue into a dispersed single-cell suspension suitable for imaging.

▶ 06:40
4
Acquire time-lapse fluorescence microscopy images

Mount dispersed cells on a microscope slide and capture fluorescent reporter oscillations using high-resolution time-lapse imaging over several hours.

▶ 07:58
5
Process and analyze time-lapse image data

Apply image registration, background subtraction, and quantitative analysis to extract cyclic gene expression signals from acquired microscopy movies.

▶ 09:18
6
Interpret single-cell oscillation patterns

Compare representative time-lapse results to identify cell-autonomous segmentation clock oscillations and synchronization dynamics in vitro.

▶ 10:00
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