Intrahepatic bile ducts are lined by biliary epithelial cells (BECs). However, defining the genetic heterogeneity of BECs remains challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize Sox9EGFP transgene expression in the liver and demonstrate that GFP expression levels are associated with distinct cell types. BECs express low or high levels of GFP, while periportal hepatocytes express sublow GFP. Sox9EGFP distribution varies by duct size, with GFPhigh BECs found at greater numbers in smaller ducts. RNA-seq reveals distinct gene expression signatures for Sox9EGFP populations and enrichment of Notch and Yap signaling in GFPlow and GFPhigh BECs. All GFP+ populations are capable of forming organoids, but demonstrate interpopulation differences in organoid survival and size, dependent on media conditions. Organoids derived from Sox9EGFP populations also demonstrate differential activation of HNF4A protein in hepatocyte media conditions, suggesting variable potency in BEC subpopulations. We find that Yap signaling is required to maintain Sox9 expression in biliary organoids, and that bile acids are insufficient to induce Yap activity or Sox9 in vivo and in vitro. Our data demonstrate that Sox9EGFP levels provide a readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.

中文翻译：

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Sox9EGFP定义Yap活动下游的胆汁上皮异质性

肝内胆管衬有胆管上皮细胞（BEC）。但是，定义BEC的遗传异质性仍然具有挑战性，并且用于识别BEC亚群的工具也很有限。在这里，我们表征肝脏中的Sox9EGFP转基因表达，并证明GFP表达水平与不同的细胞类型相关。BEC表达低水平或高水平的GFP，而门静脉肝细胞表达低水平的GFP。Sox9EGFP的分布因导管大小而异，在较小导管中，GFP高BEC数量较多。RNA-seq揭示了Sox9EGFP群体的独特基因表达特征以及GFPlow和GFPhigh BEC中Notch和Yap信号的富集。所有GFP +种群都能形成类器官，但根据培养基条件，种群间的类器官存活率和大小存在差异。源自Sox9EGFP群体的类器官在肝细胞培养基条件下还表现出HNF4A蛋白的差异激活，表明BEC亚群的可变能力。我们发现需要Yap信号来维持胆汁类器官中Sox9的表达，并且胆汁酸不足以在体内和体外诱导Yap活性或Sox9。我们的数据表明，Sox9EGFP水平可提供Yap活性的读数并描绘BEC异质性，从而为检测肝脏中亚群特异性细胞功能提供了一种工具。胆汁酸不足以在体内和体外诱导Yap活性或Sox9。我们的数据表明，Sox9EGFP水平可提供Yap活性的读数并描绘BEC异质性，从而为检测肝脏中亚群特异性细胞功能提供了一种工具。胆汁酸不足以在体内和体外诱导Yap活性或Sox9。我们的数据表明，Sox9EGFP水平可提供Yap活性的读数并描绘BEC异质性，从而为检测肝脏中亚群特异性细胞功能提供了一种工具。