Instituto de Medicina Molecular and Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa, Av Prof, Egas Moniz, 1649-028 Lisboa, Portugal.
BMC Biol. 2011 Aug 31;9:58. doi: 10.1186/1741-7007-9-58.
Building the complex vertebrate nervous system involves the regulated production of neurons and glia while maintaining a progenitor cell population. Neurogenesis starts asynchronously in different regions of the embryo and occurs over a long period of time, allowing progenitor cells to be exposed to multiple extrinsic signals that regulate the production of different cell types. Notch-mediated cell-cell signalling is one of the mechanisms that maintain the progenitor pool, however, little is known about how the timing of Notch activation is related to the cell cycle and the distinct modes of cell division that generate neurons. An essential tool with which to investigate the role of Notch signalling on cell by cell basis is the development a faithful reporter of Notch activity.
Here we present a novel reporter for Notch activity based on the promoter of the well characterised Notch target chick Hes5-1, coupled with multiple elements that confer instability, including a destabilized nuclear Venus fluorescent protein and the 3' untranslated region (UTR) of Hes5-1. We demonstrate that this reporter faithfully recapitulates the endogenous expression of Hes5-1 and that it robustly responds to Notch activation in the chick neural tube. Analysis of the patterns of Notch activity revealed by this reporter indicates that although Notch is most frequently activated prior to mitosis it can be activated at any time within the cell cycle. Notch active progenitors undergoing mitosis generate two daughters that both continue to experience Notch signalling. However, cells lacking Notch activity before and during mitosis generate daughters with dissimilar Notch activity profiles.
A novel Notch reporter with multiple destabilisation elements provides a faithful read-out of endogenous Notch activity on a cell-by-cell basis, as neural progenitors progress through the cell cycle in the chick neural tube. Notch activity patterns in this cell population provide evidence for distinct Notch signalling dynamics underlying different cell division modes and for the involvement of random initiation of Notch signalling within the neuroepithelium. These findings highlight the importance of single-cell analysis in the study of the complexity of Notch activity and provide new insights into the mechanisms underlying cell fate decisions in neural progenitors.
构建复杂的脊椎动物神经系统需要调节神经元和神经胶质的产生，同时维持祖细胞群体。神经发生在胚胎的不同区域异步开始，并持续很长一段时间，使祖细胞能够暴露于多种调节不同细胞类型产生的外源性信号中。Notch 介导的细胞间信号是维持祖细胞库的机制之一，然而，关于 Notch 激活的时间如何与细胞周期以及产生神经元的不同细胞分裂模式相关知之甚少。研究 Notch 信号对细胞的作用的一个重要工具是开发 Notch 活性的忠实报告基因。
在这里，我们提出了一种基于特征明确的 Notch 靶标 chick Hes5-1 启动子的 Notch 活性的新型报告基因，与赋予不稳定性的多个元件结合，包括不稳定的核 Venus 荧光蛋白和 Hes5-1 的 3'非翻译区 (UTR)。我们证明，该报告基因忠实地再现了 Hes5-1 的内源性表达，并且它在鸡神经管中对 Notch 激活有强烈的反应。对该报告基因揭示的 Notch 活性模式的分析表明，尽管 Notch 通常在有丝分裂前被激活，但它可以在细胞周期的任何时间被激活。进行有丝分裂的 Notch 活性祖细胞产生两个继续经历 Notch 信号的子细胞。然而，在有丝分裂前和有丝分裂期间缺乏 Notch 活性的细胞产生具有不同 Notch 活性谱的子细胞。
带有多个不稳定性元件的新型 Notch 报告基因为鸡神经管中神经祖细胞通过细胞周期提供了内源性 Notch 活性的忠实读出。该细胞群体中的 Notch 活性模式为不同细胞分裂模式下不同的 Notch 信号动力学以及神经上皮中 Notch 信号的随机起始提供了证据，并为神经祖细胞中的细胞命运决定提供了新的见解。这些发现强调了单细胞分析在 Notch 活性复杂性研究中的重要性，并为神经祖细胞中细胞命运决定的机制提供了新的见解。
