High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript’s level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a ‘core’ module of antiviral genes is expressed very early by a few ‘precocious’ cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced ‘peaked’ inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses. Large-scale single-cell RNA-seq of stimulated primary mouse bone-marrow-derived dendritic cells highlights positive and negative intercellular signalling pathways that promote and restrain cellular variation. High-throughput single-cell transcriptomics offers an unbiased approach for understanding gene expression variation between cells. Here, Aviv Regev and colleagues present single-cell RNA-seq libraries obtained from primary mouse bone-marrow-derived dendritic cells subjected to diverse perturbations — including stimulation of individual cells in isolated, sealed microfluidic chambers and genetic and chemical alterations of paracrine signalling. The results show how the antiviral and inflammatory response modules of dendritic cells are controlled by positive and negative intercellular paracrine feedback loops that both promote and restrain variation.

Single-cell RNA-seq reveals dynamic paracrine control of cellular variation