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.
Relevance to Public Health: The Notch signaling pathway enables cell-cell communication in critical and diverse developmental processes including neurogenesis, somitogenesis, angiogenesis, and hematopoietic development, and is implicated in many diseases, making Notch a major therapeutic target. Using quantitative single-cell analysis of reconstituted signaling pathways together with mathematical modeling, this proposal will provide an understanding of how molecular components of the Notch pathway function together through a newly discovered dynamic ?code? that ensures that the correct target gene expression programs are activated in individual cells. These studies will elucidate fundamental mechanisms of Notch function relevant for diverse diseases and pharmacological interventions.
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| LeBon, Lauren; Lee, Tom V; Sprinzak, David et al. (2014) Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states. Elife 3:e02950 |
