A novel reporter of notch signalling indicates regulated and random Notch activation during vertebrate neurogenesis.
A novel reporter of notch signalling indicates regulated and random Notch activation during vertebrate neurogenesis.
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DOI：
10.1186/1741-7007-9-58
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发表时间：
2011-08-31
期刊：
影响因子：
5.4
通讯作者：
Henrique D
中科院分区：
文献类型：
--
作者：
Vilas-Boas F;Fior R;Swedlow JR;Storey KG;Henrique D
构建复杂的脊椎动物神经系统涉及神经元和神经胶质的调节生产，同时保持祖细胞群。神经发生在胚胎的不同区域异步开始，并在很长一段时间内发生，使祖细胞暴露于多种调节不同细胞类型产生的外来信号。Notch介导的细胞-细胞信号传导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和产生神经元的细胞分裂的不同模式相关知之甚少。研究Notch信号传导在逐个细胞基础上的作用的一个重要工具是开发Notch活性的忠实报告者。在这里，我们提出了一种新的Notch活性的报告基因，该基因基于Notch靶鸡Hes 5 -1的启动子，加上多个赋予不稳定性的元件，包括不稳定的核Venus荧光蛋白和Hes 5 -1的3'非翻译区（UTR）。我们证明，该报告忠实地再现了内源性表达的Hes 5 -1，它强烈响应于鸡神经管中的Notch激活。对该报告者揭示的Notch活性模式的分析表明，尽管Notch在有丝分裂之前最频繁地被激活，但它可以在细胞周期内的任何时间被激活。进行有丝分裂的Notch活性祖细胞产生两个子细胞，这两个子细胞都继续经历Notch信号传导。然而，在有丝分裂之前和期间缺乏Notch活性的细胞产生具有不同Notch活性谱的子代。具有多个去稳定元件的新型Notch报告基因在逐个细胞的基础上提供了内源性Notch活性的忠实读出，因为神经祖细胞在鸡神经管中通过细胞周期进展。Notch活性模式在这个细胞群体中提供的证据，不同的Notch信号转导动力学的基础上不同的细胞分裂模式和参与随机启动的Notch信号在神经上皮细胞。这些发现强调了单细胞分析在研究Notch活性复杂性中的重要性，并为神经祖细胞细胞命运决定的潜在机制提供了新的见解。
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.
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