Single Cell transcriptomes highlight the temporal hierarchy of fate decisions of the developmental tree from Neural Border Stages to Migration of NC Cells.

Gene regulatory networks govern the steps of embryonic development and control cell transcriptional states. The neural crest cells are an archetypal example of cellular diversification from a multipotent progenitor population. However, the full sequence of molecular choices governing the emergence of neural crest heterogeneity from the ectoderm state remains elusive. Here we combine ultra-dense single cell transcriptomes with machine-learning strategies and experimental validation to provide a comprehensive gene regulatory network driving vertebrate neural crest fate diversification from induction to early migration stages. Transcription factor connectome and bifurcation analyses discover that several early neural crest fates emerge at the neural plate stage, alongside an unbiased multipotent neural crest lineage persisting until after epithelial-mesenchymal transition. We also define a new and transient neural border zone state, preceding choice between neural crest and placodes during gastrulation. Our comprehensive experimental approach combined to several ML strategies broadly applicable to single cell transcriptomics deciphers the circuits driving cranial and vagal neural crest formation and provides a general model for investigating vertebrate GRNs in development, evolution and disease.