Summary Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.
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