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的决定，起着关键作用 在多种疾病中发挥作用，并代表着一个主要的药物靶点。该途径使用多个配体和 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。我们实验室最近的研究表明，这些相互作用包括 不同的配体激活不同的目标程序，甚至通过 同样的受体。代码似乎通过动态编码机制运行，其中不同的编码机制 配体以脉动或持续的方式激活Notch受体。这些不同的动态依次 有选择地激活不同的目标程序。目前对该代码的理解仅限于两种 配基和一个受体。通过阐明完整的密码，涵盖所有配体-受体-条纹组合， 我们将实现对具有任意Notch的细胞之间信号相互作用的定量预测 不同发育和疾病背景下的成分表达谱。破译这一点 代码及其功能角色，我们将结合细胞系工程，定量单细胞延时 成像，使用突变受体和药理学干扰直接控制Notch动力学，以及 神经干细胞和鸡胚的Notch动力学分析。在​Aim 1​​中，我们将映射动态 通过Notch受体、配体和边缘蛋白的全基质的信号模式(脉冲性或持续性) 组合。在​Aim 2​​中，我们将通过确定受体是否为 细胞内结构域的组成影响靶基因的表达，或者是否具有靶程序的特异性 仅取决于信号的强度和动态。最后，我们将分析Notch的功能 鸡脊髓发育的动态代码(​Aim 3​​)。具体地说，我们将使用荧光记者 由Notch独家激活，加上一种新的组织切片制剂，可以对 脊髓发育过程中的单个活细胞，将Notch动力学与细胞命运决定联系起来。 总而言之，这些结果将揭示Notch底层的动态代码的结构和功能 信号，并展示它是如何在中央脊椎动物的发育过程中运作的。

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.