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AbstractPrecise control of developmental processes is encoded in the genome in the form of gene regulatory networks (GRNs). Such multi-factorial systems are difficult to decode in vertebrates owing to their complex gene hierarchies and transient dynamic molecular interactions. Here we present a genome-widein vivoreconstruction of the GRN underlying development of neural crest (NC), an emblematic embryonic multipotent cell population. By coupling NC-specific epigenomic and single-cell transcriptome profiling with genome/epigenome engineeringin vivo, we identify multiple regulatory layers governing NC ontogeny, including NC-specific enhancers and super-enhancers, noveltrans-factors andcis-signatures. Assembling the NC regulome has allowed the comprehensive reverse engineering of the NC-GRN at unprecedented resolution. Furthermore, identification and dissection of divergent upstream combinatorial regulatory codes has afforded new insights into opposing gene circuits that define canonical and neural NC fates. Our integrated approach, allowing dissection of cell-type-specific regulatory circuitsin vivo, has broad implications for GRN discovery and investigation.

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