A new model of Notch signaling: Control of Notch receptor cis-inhibition via Notch ligand dimers
All tissue development and replenishment relies upon the breaking of symmetries leading to the morphological and operational differentiation of progenitor cells into more specialized cells. One of the main engines driving this process is the Notch signal transduction pathway, a ubiquitous signalling system found in the vast majority of metazoan cell types characterized to date. Broadly speaking, Notch receptor activity is governed by a balance between two processes: 1) intercellular Notch transactivation triggered via interactions between receptors and ligands expressed in neighbouring cells; 2) intracellular cis inhibition caused by ligands binding to receptors within the same cell. Additionally, recent reports have also unveiled evidence of cis activation. Whilst context-dependent Notch receptor clustering has been hypothesized, to date, Notch signalling has been assumed to involve an interplay between receptor and ligand monomers. In this study, we demonstrate biochemically, through a mutational analysis of DLL4, both in vitro and in tissue culture cells, that Notch ligands can efficiently self-associate. We found that the membrane proximal EGF-like repeat of DLL4 was necessary and sufficient to promote oligomerization/dimerization. Mechanistically, our experimental evidence supports the view that DLL4 ligand dimerization is specifically required for cis-inhibition of Notch receptor activity. To further substantiate these findings, we have adapted and extended existing ordinary differential equation-based models of Notch signalling to take account of the ligand dimerization-dependent cis-inhibition reported here. Our new model faithfully recapitulates our experimental data and improves predictions based upon published data. Collectively, our work favours a model in which net output following Notch receptor/ligand binding results from ligand monomer-driven Notch receptor transactivation (and cis activation) counterposed by ligand dimer-mediated cis-inhibition. Author summaryThe growth and maintenance of tissues is a fundamental characteristic of metazoan life, controlled by a highly conserved core of cell signal transduction networks. One such pathway, the Notch signalling system, plays a unique role in these phenomena by orchestrating the generation of the phenotypic and genetic asymmetries which underlie tissue development and remodeling. At the molecular level, it achieves this via two specific types of receptor/ligand interaction: intercellular binding of receptors and ligands expressed in neighbouring cells, which triggers receptor activation (transactivation); intracellular receptor/ligand binding within the same cell which blocks receptor activation (cis inhibition). Together, these counterposed mechanisms determine the strength, the direction and the specificity of Notch signalling output. Whilst, the basic mechanisms of receptor transactivation have been delineated in some detail, the precise nature of cis inhibition has remained enigmatic. Through a combination of experimental approaches and computational modelling, in this study, we present a new model of Notch signalling in which ligand monomers promote Notch receptor transactivation, whereas cis inhibition is induced optimally via ligand dimers. This is the first model to include a concrete molecular distinction, in terms of ligand configuration, between the main branches of Notch signalling. Our model faithfully recapitulates both our presented experimental results as well as the recently published work of others, and provides a novel perspective for understanding Notch-regulated biological processes such as embryo development and angiogenesis.