Combinatorial BMP4 and Activin direct choice between alternate routes to endoderm during human gastrulation [RN25025_RNAseq]

Cellular differentiation requires the proper interpretation of multiple signalling cues which vary in concentration. How exactly the combination and history of signals a cell is exposed to influences a fate decision remains poorly understood. In this study we use hESCs as a tractable model system to explore how combinations of cues guide state transitions during gastrulation. Using single-cell transcriptomics and live-cell imaging of engineered hESCs expressing endogenous cell state reporters, we reconstructed developmental lineages and obtained single-cell measurements of fate specification dynamics during gastrulation. We found that definitive endoderm, one of the three germ layers, arises from two distinct developmental trajectories: a direct route from pluripotency, and an indirect route via a mesoderm progenitor state. Furthermore, by modulating the signalling input we found that the relative concentration of Activin and BMP4 controls the choice between alternate trajectories to endoderm. These findings reveal a lineage convergence event during human gastrulation with multiple routes existing to definitive endoderm dictated by the combination of signalling cues presented. This work shows that the combination cues a cell is exposed to not only directs the final fate outcome it assumes, but the developmental route taken. The goal of this study was to characterise the transcriptional differences between definitive endoderm cells derived from two distinct developmental trajectories. This was achieved by ultilising a H1 hESCs (human embryonic stem cells) engineered with a FOXC1 Achilles transcriptional reporter. After 72 hours of differentiation in response to Activin 100 ng/mL and BMP4 10 ng/mL hESCs were fixed with 4% PFA, stained for SOX17 and sorted into four disitnct populations: SOX17+ Achilles+, SOX17+ Achilles-, SOX17- Achilles+ and SOX17+ Achilles+ and RNA extracted for sequencing. Experiments were performed in triplicate. Control cells were maintained in mTeSR1.