Deciphering the Dynamic Notch Signaling Code

摘要 Notch信号通路在不同组织环境中指导细胞命运决定，在疾病中起关键作用， 并且是主要的药物靶点。该途径使用多个配体和受体， 另一种以混杂的方式，以及调节这些相互作用的边缘糖基转移酶。 我们实验室最近的工作表明，这些相互作用构成了一个通信“代码”，其中不同的 配体以不同的动力学激活Notch受体。这些动态反过来又被解码， 激活不同的转录靶程序。目前对密码的理解仅限于两种配体 一个受体。确定动态编码如何在更广泛的 配体-受体-边缘的组合将使人们能够更好地理解、预测和控制信号传导 不同细胞类型在不同发育和疾病背景下的相互作用。在这里，我们将联合收割机 细胞系工程，定量单细胞延时成像，使用突变体直接控制Notch动力学 受体和药理学扰动，以及神经干细胞和鸡中Notch动力学分析 胚胎来破译这种代码及其功能作用。在目标1中，我们将重点关注编码，通过映射 跨越Notch受体、配体和Fringe蛋白组合的全矩阵的动态信号传导模式。我们 将进一步扩展这种方法，以分析同一细胞中多种Notch受体的共表达， 在自然细胞类型中经常发生的模式。在目标2中，我们将专注于解码，通过计算和 实验研究顺式调节和反式调节机制如何共同实现不同的 信号传导动力学以选择性地激活不同的靶基因表达程序。最后，在目标3中， 利用小鼠神经干细胞和鸡胚，分析Notch动态密码子在神经发生中的作用 胚胎脊髓发育作为模型系统。在神经干细胞中， 共培养，延时显微镜，和终点多重单分子RNA-FISH，我们将映射 配体-受体组合与Notch活性动力学，并将这些动力学反过来与细胞命运相关 决策在鸡胚中，Notch特异性荧光报告基因与新的组织切片制备物一起， 能够在脊髓发育过程中对单个活细胞进行成像，将使我们能够将Notch 细胞命运决定的动力学总之，这些结果将揭示的结构和功能， 动态代码底层Notch信号，并显示它如何在关键的发展背景下运作。更 一般来说，它们应该有助于建立一个理解信号编码和解码行为的范例， 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.