ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

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 a ntibody and m R NA t argeting seq uential f luorescence i n s itu h ybridization (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.