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Cell Biology JoVE (Open Access) Citable · DOI

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

DOI: 10.3791/55127-v
What you'll learn
  • ✓ Dissect and isolate pre-somitic mesoderm (PSM) explants from vertebrate embryos
  • ✓ Apply immunohistochemistry and FISH to detect oscillatory gene expression patterns
  • ✓ Extract temporal dynamics from spatial expression data using computational analysis
  • ✓ Interpret spatiotemporal expression profiles of Notch signaling components in segmentation
Protocol

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Difficulty
advanced
Total time
~3–4 days (embryo collection, dissection, IHC/FISH staining, imaging, computational analysis)
Model organism
Vertebrate embryo (species not specified in abstract; likely mouse or chicken)
Biosafety
BSL-1

Steps

1
Dissect pre-somitic mesoderm explants

Isolate and extract PSM tissue from embryonic explants under a dissecting microscope. This tissue retains the segmentation clock for temporal gene expression analysis.

▶ 00:50
2
Perform immunohistochemistry on PSM explants

Apply antibodies to detect protein expression (e.g., Notch receptor and Delta ligand) in fixed PSM tissue sections. This identifies spatial distribution of signaling molecules.

▶ 02:49
3
Conduct fluorescent in situ hybridization

Use FISH probes to detect mRNA expression of clock genes (e.g., Lfng, Dll1, Notch1) in PSM explants. This reveals temporal oscillation patterns across tissue regions.

▶ 04:38
4
Prepare samples for fluorescence imaging

Mount and prepare stained PSM explants on slides or imaging chambers to capture high-resolution spatial expression maps.

▶ 07:09
5
Analyze spatiotemporal expression profiles

Apply computational methods to extract temporal ordering and oscillatory dynamics from spatial expression data, correlating Delta/Notch expression with clock gene (Lfng) phases.

▶ 08:33
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