SummaryGene expression levels vary greatly within similar cells, even within clonal cell populations [1]. These spontaneous expression differences underlie cell fate diversity in both differentiation and disease [2]. The mechanisms responsible for generating expression variability are poorly understood. Using single-cell transcriptomics, we show that transcript variability emerging during Dictyostelium differentiation is driven predominantly by repression rather than activation. The increased variability of repressed genes was observed over a broad range of expression levels, indicating that variability is actively imposed and not a passive statistical effect of the reduced numbers of molecules accompanying repression. These findings can be explained by a simple model of transcript production, with expression controlled by the frequency, rather than the magnitude, of transcriptional firing events. Our study reveals that the generation of differences between cells can be a direct consequence of the basic mechanisms of transcriptional regulation.

The generation of multiple fates from a uniform cell population via selforganisation is a recurring feature in development and regeneration. However, for most self-organising systems, we have little understanding of the processes that allow cells to become different. One of the clearest examples of developmental self-organisation is shown by Dictyostelium, with cells segregating into two major fates, stalk and spore, within multicellular aggregates. To characterise the gene expression decisions that underlie this cell fate bifurcation, we carried out single cell transcriptomics on Dictyostelium aggregates. Our data show the transition of progenitors into prespore and prestalk cells occurs via distinct developmental intermediates. Few cells were captured switching between states, with minimal overlap in fate marker expression between cell types, suggesting states are discrete and transitions rapid. Surprisingly, fate-specific transcript dynamics were a small proportion of overall gene expression changes, with transcript divergence coinciding precisely with large-scale remodelling of the transcriptome shared by prestalk and prespore cells. These observations suggest the stepwise separation of cell identity is temporally coupled to global expression transitions common to both fates.

Stimulation of the ERK/MAPK pathway is required for the exit from pluripotency and onset of differentiation in mouse embryonic stem cells (ESCs). The dynamic behaviour of ERK activity in individual cells during this transition is unclear. Using a FRET-based biosensor, we monitored ERK signalling dynamics of single mouse ESCs during differentiation. ERK activity was highly heterogeneous, with considerable variability in ERK signalling between single cells within ESC colonies. Different triggers of differentiation induced distinct ERK activity profiles. Surprisingly, the dynamic features of ERK signalling were not strongly coupled to loss of pluripotency marker expression, regardless of the differentiation stimulus, suggesting the normal dynamic range of ERK signalling is not rate-limiting in single cells during differentiation. ERK signalling dynamics were sensitive to the degree of cell crowding and were similar in neighbouring cells. Sister cells from a mitotic division also showed more similar ERK activity, an effect that was apparent whether cells remained adjacent or moved apart after division. These data suggest a combination of cell lineage and niche contributes to the absolute level of ERK signalling in mouse ESCs.