A high-resolution protein atlas is essential for understanding the molecular basis of biological processes.Using protein-fusion reporters and imaging-based single-cell analyses, we present a protein expression atlas of C. elegans embryogenesis encompassing 266 transcription factors (TFs) in nearly all (90%) lineage-resolved cells. Single-cell analysis reveals a combinatorial code and cascade that elucidate the regulatory hierarchy between a large number of lineage-, tissue-, and time-specific TFs in spatiotemporal fate patterning. Guided by expression, we identify essential functions of CEH-43/DLX, a lineage-specific TF, and ELT-1/GATA3, a well-known skin fate specifier, in neuronal specification; and M03D4.4 as a pan-muscle TF in converging muscle differentiation in the body wall and pharynx. Finally, systems-level analysis of TF regulatory state uncovers lineage-and time-specific kinetics of fate progression and widespread detours of the trajectories of cell differentiation. Collectively, our work reveals a single-cell molecular atlas and general principles underlying the spatiotemporal patterning of a metazoan embryo.

Highlights d Quantification of cell position variability during C. elegans embryogenesis d Positional variability is highly deterministic and temporally dynamic d Positional variability is associated with lineage identity, location, and symmetry d Fate specification and cell-cell junctions contribute to reduction of variability