Lineage specification requires accurate interpretation of multiple signaling cues. However, how combinatorial signaling histories influence fate outcomes remains unclear. We combined single-cell transcriptomics, live-cell imaging, and mathematical modeling to explore how activin and bone morphogenetic protein 4 (BMP4) guide fate specification during human gastrulation. We see that these signals interact both synergistically and antagonistically to drive fate decisions. We find that definitive endoderm arises from lineage convergence: a direct route from pluripotency and an indirect route via a mesoderm progenitor state. Cells pass through temporal windows of signaling competency, and the relative concentration of activin and BMP4 dictates the trajectory choice. The efficiency between routes is underpinned by a dual role of BMP4 in inducing mesoderm genes while promoting pluripotency exit. This work underscores that the combination of signals a cell is exposed to not only directs its final fate but also the developmental route taken, suggesting lineage convergence enhances robustness in fate specification.
Keywords: BMP4; activin; combinatorial signaling; endoderm; fate specification; hESC; human gastrulation; modeling; scRNA-seq; state transitions.
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