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.

The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes.

Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.