Elucidating the Dynamic Code of the Notch Signaling Pathway

摘要 Notch信号通路在几乎所有组织的发育中指导细胞命运决定， 在许多疾病中起作用，并且是主要的药物靶点。该途径使用多种配体， 以混杂方式相互作用的受体，以及边缘糖基转移酶 来调节这些相互作用。我们实验室最近的工作表明，这些相互作用包括 不同的配体激活不同的目标程序的通信“代码”，甚至通过 相同的受体。代码似乎通过动态编码机制运行，其中不同的 配体以脉冲或持续方式激活Notch受体。这些不同的动力反过来 选择性地激活不同的目标程序。目前对代码的理解仅限于两个 配体和一个受体。通过阐明完整的密码，涵盖所有配体-受体-边缘组合， 我们将能够定量预测具有任意Notch的细胞之间的信号相互作用， 不同发育和疾病背景下的组分表达谱。为了破译这个 代码及其功能作用，我们将联合收割机结合细胞系工程，定量单细胞时间推移 成像，使用突变受体和药理学扰动直接控制Notch动力学，以及 分析神经干细胞和鸡胚中的Notch动力学。在目标1中，我们将动态映射 跨越Notch受体、配体和Fringe蛋白的完整基质的信号传导模式（脉冲或持续） 组合。在目标2中，我们将通过确定受体是否 胞内结构域组成影响靶基因表达，或靶程序特异性 只取决于信号的强度和动态。最后，我们将分析Notch的功能 鸡脊髓发育中的动态代码（Aim 3）。具体来说，我们将使用荧光报告器 由Notch专门激活，以及一种新的组织切片制备， 脊髓发育过程中的单个活细胞，以将Notch动力学与细胞命运决定联系起来。 总之，这些结果将揭示Notch底层动态代码的结构和功能 信号，并显示它是如何运作的一个中央脊椎动物的发展过程。

Abstract The Notch signaling pathway directs cell fate decisions in development of nearly every tissue, plays key roles in numerous diseases, and represents a major drug target. The pathway uses multiple ligands and receptors that interact with one another in a promiscuous fashion, as well as Fringe glycosyltransferases that modulate those interactions. Recent work from our lab has revealed that these interactions comprise a communication ‘code’ in which different ligands activate different target programs, even through the same receptors. The code appears to function through a dynamic encoding mechanism, in which different ligands activate Notch receptors in either a pulsatile or sustained fashion. These different dynamics in turn selectively activate distinct target programs. Current understanding of the code is limited to just two ligands and one receptor. By elucidating the full code, covering all ligand-receptor-Fringe combinations, we will enable quantitative prediction of signaling interactions between cells with arbitrary Notch component expression profiles across different developmental and disease contexts. To decipher this code and its functional roles, we will combine cell line engineering, quantitative single-cell time-lapse imaging, direct control of Notch dynamics using mutant receptors and pharmacological perturbations, and analysis of Notch dynamics in neural stem cells and chick embryos. In ​Aim 1​​, we will map dynamic signaling modes (pulsatile or sustained) across a full matrix of Notch receptor, ligand, and Fringe protein combinations. In ​Aim 2​​, we will analyze dynamic signal decoding by determining whether receptor intracellular domain composition affects target gene expression, or whether target program specificity depends only on the strength and dynamics of signaling. Finally, we will analyze the function of the Notch dynamic code in chick spinal cord development (​Aim 3​​). Specifically, we will use a fluorescent reporter exclusively activated by Notch, together with a new tissue slice preparation that enables imaging of individual living cells during spinal cord development, to link Notch dynamics to cell fate determination. Together, these results will reveal the structure and function of the dynamic code underlying Notch signaling, and show how it operates in a central vertebrate developmental process.