Consecutive waves of NODAL inhibition are required for epiblast patterning

The mouse genome encodes two Lefty genes that antagonize NODAL signaling to break symmetry during gastrulation. Despite their central regulatory role, accurately describing and comparing their embryonic function remains unclear. Here, we generated high replicate powered single-cell transcriptional atlases of Lefty1 and Lefty2 mutant embryogenesis to evaluate the effects of perturbed NODAL signaling during gastrulation. We find that both mutants exhibit phenotypes beginning with the earliest primitive streak derivatives, suggesting a sequential shift from anterior Lefty1 to posterior Lefty2-based control that prevents spurious differentiation into extraembryonic and axial mesoendoderm at the cost of embryonic mesoderm and, in the case of Lefty1, rostral forebrain specification. By phasing bulk SMAD2/3 ChIP-seq data into single-cell-resolved descriptions of signal transduction, we identify distinct NODAL-responsive modules that track the context-specific use of this morphogen over gastrulation time. Collectively, our work highlights the potential of single-cell assays to decode how complex differentiation processes resolve morphogen signals in space and time.