SRP327837 PRJNA565680 GSE137462 Early chromatin shaping predetermines multipotent vagal neural crest into neural, neuronal and mesenchymal lineages [scRNA-seq] The enteric nervous system (ENS) predominantly originates from vagal neural crest cells (VNC) that emerge from the caudal hindbrain, invade the foregut and populate the gastrointestinal tract. However, the gene regulatory network (GRN) orchestrating the early specification of VNC remains unknown. Using an EdnrB enhancer, we generated a comprehensive temporal map of the chromatin and transcriptional landscape of VNC in the avian model, revealing three VNC cell clusters (neural, neurogenic and mesenchymal), each predetermined epigenetically prior to neural tube delamination. We identify and functionally validate regulatory cores (Sox10/Tfap2B/SoxB/Hbox) mediating each programme and elucidate their combinatorial activities with other spatiotemporally-specific transcription factors (bHLH/NR). Our global deconstruction of the VNC-GRN in vivo sheds light on critical early regulatory mechanisms that may influence the divergent neural phenotypes in enteric neuropathies. Overall design: Single neural crest cells isolated from HH18 avian embryo using FACS, single cell RNA-seq performed by 10X GSE137462 pubmed 31792380 pubmed 33111104 parent_bioproject PRJNA517324