Spatial modelling of NOTCH signalling predicts tuneable dynamic heterogeneity in endothelia.

Endothelial cells (ECs) are phenotypically heterogeneous to perform specialised functions across different organs and tissues. Although environmental inputs are considered key drivers, we previously observed persistent diversity in NOTCH pathway activation within quiescent endothelia, but the underlying mechanism remains unclear. Here, we developed a computational-experimental framework linking core NOTCH dynamics to emergent EC heterogeneity in space and time. We built an agent-based model of NOTCH signalling incorporating lateral inhibition, lateral induction, cis interactions, and optional HES1 autoregulation, and constrained timescales using HUVEC cultures under γ-secretase inhibition. Model exploration identified regimes with emergent asynchronous HES1 oscillations driven by productive cis interactions or by autoregulation. Contact-modulated cis/trans interactions reproduced the experimentally observed spatial distributions, whereas autoregulation produced globally synchronised dynamics inconsistent with spatial heterogeneity. Additionally, oscillation amplitude and local synchrony were tuneable by varying biologically relevant parameters. Consistent with these predictions, sparse-versus-confluent experiments showed that intercellular contact is required for spatial heterogeneity. Overall, our results support the hypothesis that intrinsic, contact-dependent NOTCH dynamics can generate heterogeneous EC phenotypes, providing a mechanistic basis for functional plasticity in quiescent monolayers. We provide the calibrated model as an open-source, extensible resource for reproducible testing of alternative NOTCH interaction hypotheses against future quantitative datasets.