Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping
Gehart, Helmuth; van Es, Johan H.; Hamer, Karien; Beumer, Joep; Kretzschmar, Kai; Dekkers, Johanna F.; Rios, Anne; Clevers, Hans
(2019) Cell, volume 176, issue 5, pp. 1158 - 1173.e16
(Article)
Abstract
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
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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. The hierarchical lineage of intestinal enteroendocrine cells is defined at a spatiotemporal single-cell manner and validated using organoid and in vivo models.
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Keywords: Animals, Basic Helix-Loop-Helix Transcription Factors/genetics, Cell Differentiation/genetics, Cell Lineage/genetics, Enteroendocrine Cells/metabolism, Fluorescent Dyes, Gene Expression Profiling/methods, Homeodomain Proteins/genetics, Intestinal Mucosa/cytology, Mice, Mice, Knockout, Nerve Tissue Proteins/genetics, Optical Imaging/methods, Organoids, Phenotype, Single-Cell Analysis/methods, Stem Cells, Transcription Factors/genetics, Transcriptome/genetics, Journal Article, Research Support, Non-U.S. Gov't
ISSN: 0092-8674
Publisher: Cell Press
Note: Funding Information: We thank Stefan vd Elst and Reinier vd Linden for flow cytometry assistance, Harry Begthel for immunohistochemistry help, Judith Vivié for SORT-seq support, the Utrecht Sequencing Facility (USF) and Genome Scan for sequencing, Anna Alemany Arias and Alexander van Oudenaarden for mapping RNA velocity data, Anko de Graaff and the Hubrecht Imaging Centre (HIC) for microscopy assistance, Jeroen Korving for help with mouse generation, Cayetano Pleguezuelos Manzano for experimental help, and Zoraide Granchi for careful reading of the manuscript. Tac1Cre-LSL-tdTomato organoids were kindly provided by Hongkui Zeng. This work was supported by an EMBO long-term fellowship ( ALTF 240-2014 to H.G.) and a VENI grant from the Netherlands Organization for Scientific Research ( NWO-ZonMW, 016.166.119 to H.G.) as well as an HFSP fellowship ( LT771/2015 to K.K.) and VENI fellowship ( NWO-ZonMW, 016.166.140 to K.K.). Publisher Copyright: © 2018 Elsevier Inc. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
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