Temporal ordering of dynamic expression data from detailed spatial expression maps

Charlotte S. L. Bailey (Lead / Corresponding author), Robert A. Bone, Philip J. Murray, J. Kim Dale (Lead / Corresponding author)

Research output: Contribution to journalArticle

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Abstract

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.
Original languageEnglish
Article numbere55127
Pages (from-to)1-8
Number of pages8
JournalJoVE: Journal of Visualized Experiments
Issue number120
DOIs
Publication statusPublished - 9 Feb 2017

Fingerprint

Mesoderm
Clocks
Notch Receptors
Genes
Ligands
Periodicity
Gene expression
Gene Components
Proteins
Gene Expression
Somites
Messenger RNA
Phosphorylation
Wnt Signaling Pathway
Fibroblast Growth Factors
Fibroblasts
Image analysis
Coloring Agents
Visualization
Chemical activation

Keywords

  • Notch
  • segmentation
  • Delta
  • chick
  • mouse
  • embryo
  • oscillations

Cite this

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title = "Temporal ordering of dynamic expression data from detailed spatial expression maps",
abstract = "During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the {"}segmentation clock,{"} acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as {"}kymographs,{"} for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.",
keywords = "Notch, segmentation, Delta, chick, mouse, embryo, oscillations",
author = "Bailey, {Charlotte S. L.} and Bone, {Robert A.} and Murray, {Philip J.} and Dale, {J. Kim}",
note = "This work was supported by an MRC studentship to RAB, an MRC studentship to CSLB, and a WT project grant to JKD (WT089357MA). The work was also supported by a Wellcome Trust Strategic award (097945/Z/11/Z).",
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Temporal ordering of dynamic expression data from detailed spatial expression maps. / Bailey, Charlotte S. L. (Lead / Corresponding author); Bone, Robert A.; Murray, Philip J.; Dale, J. Kim (Lead / Corresponding author).

In: JoVE: Journal of Visualized Experiments, No. 120, e55127, 09.02.2017, p. 1-8.

Research output: Contribution to journalArticle

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AU - Bailey, Charlotte S. L.

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