Cell fate decisions are ubiquitous and play a critical role throughout development, yet how localization impacts cellular decision making remains unclear. To identify the drivers of position-dependent fate decisions at a molecular level, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridization (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins and phosphoproteins in single cells at sub-micrometre spatial resolution. We studied 67 unique (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns, yielding quantification of both abundance and localization of mRNAs and (phospho-)proteins during the first 48 hours of differentiation. ARTseq-FISH revealed a fate decision between continued self-renewal and differentiation that relies solely on the position of each mESC on the micropattern. Our results demonstrate that temporal changes in cell cycle orchestrate these position-dependent cell fate decisions.

Co-expression of genes measured with single-cell RNA sequencing is extensively utilized to understand the principles of gene regulation within and across cell types and species. It is assumed that the presence of correlation in gene expression values at the single-cell level demonstrates the existence of common regulatory mechanisms. However, the regulatory mechanisms that should lead to observed co-expression at an mRNA level often remain unexplored. Here we investigate the relationship between processes upstream and downstream of transcription (i.e., promoter architecture and coordination, DNA contact frequencies and mRNA degradation) and pairwise gene expression correlations at an mRNA level. We identify that differences in mRNA degradation (i.e., half-life) is a pivotal source of single-cell correlations in mRNA levels independently of the presence of common regulatory mechanisms. These findings reinforce the necessity of including post-transcriptional regulation mechanisms in the analysis of gene expression in mammalian cells.