Bailey Charlotte S L, Bone Robert A, Murray Philip J, Dale J Kim
The Danish Stem Cell Center (DanStem), University of Copenhagen.
Division of Mathematics, University of Dundee.
J Vis Exp. 2017 Feb 9(120):55127. doi: 10.3791/55127.
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.
在体节发生过程中,成对的上皮性体节以渐进的方式形成,从前体节中胚层(PSM)的前端以严格的物种特异性周期出芽。该过程的周期性由一种分子振荡器调节,这种振荡器被称为“分割时钟”,作用于PSM细胞。这个时钟驱动基因表达在PSM中沿后 - 前方向的振荡模式。这些所谓的时钟基因是三条信号通路的关键组成部分:Wnt、Notch和成纤维细胞生长因子(FGF)。此外,Notch信号对于同步相邻细胞中的细胞内振荡至关重要。我们最近深入了解了这可能是如何在机制上进行调节的。在配体激活后,Notch受体被切割,释放出细胞内结构域(NICD),它移动到细胞核并调节基因表达。NICD非常不稳定,其磷酸化依赖性周转作用于限制Notch信号。已知PSM中NICD产生(和降解)的情况是振荡性的,并且类似于时钟基因的情况。我们最近报道,介导细胞间偶联的Notch受体和Delta配体在mRNA和蛋白质水平上都表现出动态表达。在本文中,我们描述了我们使用的灵敏检测方法和详细的图像分析工具,结合我们设计的计算模型,从表达周期的不同点提取并叠加表达数据。这使我们能够构建受体、配体和Notch靶标时钟基因在整个振荡周期内动态表达谱的时空图。在这里,我们描述了用于生成和培养PSM外植体的方案,以及mRNA或蛋白质染色的程序。我们还解释了随后如何对共聚焦图像进行分析并通过计算进行时间排序,以生成时钟表达快照的有序序列,以下定义为“波阵面图”,用于可视化Delta样1(Dll1)和Notch1在整个PSM中的时空表达。