Deciphering the Dynamic Notch Signaling Code

摘要 Notch信号通路在不同的组织环境中指导细胞命运的决定，在疾病中发挥关键作用， 代表着一个主要的毒品目标。该途径使用多个配体和受体与一个相互作用 另一种是杂乱的方式，以及调节这些相互作用的边缘糖基转移酶。 我们实验室最近的研究表明，这些相互作用构成了一种交流“代码”，其中不同 配体以不同的动力学激活Notch受体。这些动态又被有选择地解码为 激活不同的转录靶向程序。目前对密码的理解仅限于两个配体 和一个受体。确定如何在更广泛的曲目中进行动态编码 配体-受体-条纹组合将使人们能够更好地理解、预测和控制信号 不同发育和疾病背景下不同细胞类型之间的相互作用。在这里，我们将结合 细胞系工程、定量单细胞延时成像、使用突变的Notch动态的直接控制 神经干细胞和雏鸡的受体和药理扰动及Notch动力学分析 破译这一密码及其功能作用的胚胎。在​Aim 1​中，我们将重点介绍编码，通过映射 通过Notch受体、配体和边缘蛋白组合的全矩阵的动态信号模式。我们 将进一步扩展这种方法来分析多个Notch受体在同一细胞中的共同表达，一个 在自然细胞类型中经常出现的模式。在​Aim 2​中，我们将专注于解码，通过计算和 通过实验研究顺式调节和跨调节机制如何共同实现不同的 信号动力学，以选择性地激活不同的靶基因表达程序。最后，在​Aim 3中，我们将 利用小鼠神经干细胞和雏鸡分析Notch动态密码在神经发生中的作用 以胚胎脊髓发育为模型系统。在神经干细胞中，使用一种 共培养，时间推移显微镜，和终点多重单分子RNA-FISH，我们将绘制 配体-受体组合对Notch活性动态的影响，并将这些动态与细胞命运联系起来 决定。在鸡胚中，Notch特异的荧光报告与新的组织切片制备 这使得在脊髓发育过程中能够对单个活细胞进行成像，这将使我们能够将Notch 细胞命运决定的动力学。综上所述，这些结果将揭示 在Notch信号基础上的动态代码，并展示它如何在关键的发展背景下运行。更多 一般而言，它们应该有助于建立一个范例，用于理解 蜂窝通信系统。

Abstract The Notch signaling pathway directs cell fate decisions in diverse tissue contexts, plays key roles in disease, 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 suggests that these interactions comprise a communication ‘code’ in which different ligands activate Notch receptors with distinct dynamics. These dynamics are, in turn, decoded to selectively activate distinct transcriptional target programs. Current understanding of the code is limited to just two ligands and one receptor. Determining how dynamic encoding occurs across a broader repertoire of ligand-receptor-Fringe combinations will enable better understanding, prediction, and control of signaling interactions between different cell types in diverse developmental and disease contexts. Here, 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 to decipher this code and its functional roles. In ​Aim 1​, we will focus on encoding, by mapping dynamic signaling modes across a full matrix of Notch receptor, ligand, and Fringe protein combinations. We will further extend this approach to analyze co-expression of multiple Notch receptors in the same cell, a pattern that occurs frequently in natural cell types. In ​Aim 2​, we will focus on decoding, by computationally and experimentally investigating how cis-regulatory and trans-regulatory mechanisms together enable different signaling dynamics to selectively activate distinct target gene expression programs. Finally, in ​Aim 3 we will analyze the function of the Notch dynamic code in neurogenesis, using mouse neural stem cells and chick embryonic spinal cord development as model systems. In neural stem cells, using a combination of co-cultures, time-lapse microscopy, and end-point multiplexed single molecule RNA-FISH, we will map ligand-receptor combinations to Notch activity dynamics, and relate those dynamics in turn to cell fate decisions. In chick embryos, a Notch-specific fluorescent reporter, together with a new tissue slice preparation that enables imaging of individual living cells during spinal cord development, will enable us 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 key developmental contexts. More generally, they should help establish a paradigm for understanding signal encoding and decoding behaviors in cellular communication systems.