Timely NODAL Inhibition Driven by Consecutive Lefty1 and Lefty2 Is Required for Epiblast Patterning

The mouse genome encodes two Lefty genes, novel TGF-β superfamily members that antagonize the Nodal signaling gradient during symmetry breaking within the gastrulation-stage embryo. Despite their reported roles as modulators of this key morphogen, accurately describing how the Leftys contribute to cell fate determination is hindered due to the challenge of modeling rapid changes in embryonic complexity from static snapshots of internally developing embryos. Here, we generated two high temporal and cellular resolution transcriptional atlases of Lefty1 and Lefty2 mutant embryos and evaluated the effects of these regulators on cell fate specification using a Network Flow Model to describe the processes of gastrulation continuously. Combined with analysis of dynamic Nodal activity in wild-type and mutant embryos, we capture a sequential shift from Anterior Visceral endoderm (AVE)-based Lefty1 to primitive streak (PS)-based Lefty2 control, which our mutant data suggest act to collectively shield the developing embryo from spurious Definitive endoderm (DefEndo) and Extra-embryonic mesodermal (ExM) differentiation. We find that mutation of either Nodal antagonist converges towards a highly similar phenotype, with only the most rostral neural lineages protected in lefty2 mutant embryos as a hallmark of AVE-based patterning. Collectively, our work highlights the potential of single-cell profiling technologies to decode complex differentiation processes, including addressing how classical morphogens temporally coordinate multiple distinct transcriptional states from the same pluripotent field.