Single-cell technologies promise to uncover how transcriptional programs orchestrate complex processes during embryogenesis. Here, we apply a combination of single-cell technology and genetic analysis to investigate the dynamic transcriptional changes associated with Drosophila embryo morphogenesis at gastrulation. Our dataset encompassing the blastoderm-to-gastrula transition provides a comprehensive single-cell map of gene expression across cell lineages validated by genetic analysis. Subclustering and trajectory analyses revealed a surprising stepwise progression in patterning to transition zygotic gene expression and specify germ layers as well as uncovered an early role for ecdysone signaling in epithelial-to-mesenchymal transition in the mesoderm. We also show multipotent progenitors arise prior to gastrulation by analyzing the transcription trajectory of caudal mesoderm cells, including a derivative that ultimately incorporates into visceral muscles of the midgut and hindgut. This study provides a rich resource of gastrulation and elucidates spatially regulated temporal transitions of transcription states during the process.
