Recording morphogen signals reveals origins of gastruloid symmetry breaking

When cultured in three dimensional spheroids, mammalian stem cells can reproducibly self-organize a single anterior-posterior axis and sequentially differentiate into structures resembling the primitive streak and tailbud. Whereas the embryo’s body axes are instructed by spatially patterned extra-embryonic cues, it is unknown how these stem cell gastruloids break symmetry to reproducibly define a single anterior-posterior (A-P) axis. Here, we use synthetic gene circuits to trace how early intracellular signals predict cells’ future anterior-posterior position in the gastruloid. We show that Wnt signaling evolves from a homogeneous state to a polarized state, and identify a critical 6-hour time period when single-cell Wnt activity predicts future cellular position, prior to the appearance of polarized signaling patterns or morphology. Single-cell RNA sequencing and live-imaging reveal that early Wnt-high and Wnt-low cells contribute to distinct cell types and suggest that axial symmetry breaking is driven by sorting rearrangements involving differential cell adhesion. We further extend our approach to other canonical embryonic signaling pathways, revealing that even earlier heterogeneity in TGF(β) signaling predicts A-P position and modulates Wnt signaling during the critical time period. Our study reveals a sequence of dynamic cellular processes that transform a uniform cell aggregate into a polarized structure and demonstrates that a morphological axis can emerge out of signaling heterogeneity and cell movements even in the absence of exogenous patterning cues.