Cis Interactions between Notch and Delta Generate Mutually Exclusive Signaling States

The Notch-Delta signaling pathway enables communication between neighboring cells during development1. It plays a critical role in the formation of ‘fine-grained’ patterns, generating distinct cell fates among groups of initially equivalent neighboring cells, and in sharply delineating neighboring regions in developing tissues 2,3,4,5. The Delta ligand has been shown to have two activities: it trans-activates Notch in neighboring cells, and cis-inhibits Notch in its own cell. However, it remains unclear how Notch integrates these two activities and how the resulting system facilitates pattern formation. To address these questions, we developed a quantitative time-lapse microscopy platform for analyzing Notch-Delta signaling dynamics in individual mammalian cells. By controlling both cis- and trans-Delta levels, and monitoring the dynamics of a Notch reporter, we measured the combined cis-trans input-output relationship for the Notch-Delta system. The data revealed a striking difference between the response of Notch to trans- and cis-Delta: While the response to trans-Delta is graded, the response to cis-Delta exhibits a sharp, switch-like response at a fixed threshold, independent of trans-Delta. We developed a simple mathematical model that shows how these behaviors emerge from the mutual inactivation of Notch and Delta proteins. This interaction generates an ultrasensitive switch between mutually exclusive sending (high Delta / low Notch) and receiving (high Notch / low Delta) signaling states. At the multicellular level, this switch can amplify small differences between neighboring cells even without transcription-mediated feedback. This Notch-Delta signaling switch facilitates the formation of sharp boundaries and lateral inhibition patterns in models of development, and provides insight into previously unexplained mutant behaviors.