Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping
Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping
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DOI：
10.1016/j.cell.2018.12.029
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发表时间：
2019-02-21
期刊：
影响因子：
64.5
通讯作者：
Clevers, Hans
中科院分区：
文献类型：
--
作者：
Gehart, Helmuth;van Es, Johan H.;Clevers, Hans
肠内分泌谱系的稳态调节是一个知之甚少的过程。我们解决了肠内分泌分化过程中的转录变化，在真实的时间在单细胞水平上使用一种新的敲入等位基因的Neurog 3，主调节基因在发病时的肠内分泌规格简要表达。双荧光报告基因Neurog3Chrono可测量从肠内分泌分化开始的时间，并使单细胞转录组沿着绝对时间轴精确定位。这种方法产生了一个明确的描述的肠内分泌层次结构及其亚系，揭示了亚系之间的动力学差异，并揭示了时间依赖性的激素可塑性肠嗜铬细胞和L细胞。转录变化的时间分辨图谱预测了多种新的分子调节因子。其中9个通过小鼠中的条件性敲除或肠道类器官中的CRISPR修饰进行了验证。六种新的候选调节剂（Sox 4，Rfx6，Tox3，Myt 1，Runx1t1和Zcchc12）产生了特定的肠内分泌表型。我们的时间分辨单细胞转录图谱提供了丰富的资源来解开肠内分泌分化。
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 entero-chromaffin 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.