Background & Aims

Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9^EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types.

Methods

Sox9^EGFP BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9^EGFP mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence.

Results

BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9^EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFP^high BECs. Single Sox9^EGFP+ cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9^EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation.

Conclusions

Our data demonstrate that Sox9^EGFP levels provide 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 定义了 Yap 活动下游的胆管上皮异质性

背景与目标

确定肝内胆管上皮细胞 (BEC) 的遗传异质性具有挑战性，并且识别 BEC 亚群的工具有限。在这里，我们描述了 Sox9 ^EGFP转基因在肝脏中的表达，并证明了绿色荧光蛋白 (GFP) 的表达水平与不同的细胞类型相关。

方法

通过免疫荧光、流式细胞术和基因表达谱分析Sox9 ^{EGFP BAC 转基因小鼠，以表征 GFP 群体的体内特征。}通过荧光激活细胞分选分离来自不同 GFP 群体的单个 BEC，并在类器官形成测定中进行功能分析。肝内导管上皮作为类器官生长，并用 Yes 相关蛋白 (Yap) 抑制剂或胆汁酸处理以确定BEC中Sox9的上游调节。对Sox9 ^EGFP小鼠进行胆管结扎，并通过免疫荧光评估 GFP 表达。

结果

BEC 表达低或高水平的 GFP，而门静脉周围肝细胞表达亚低水平的 GFP。Sox9 ^EGFP+ BEC 因导管大小而分布不同，并表现出不同的基因表达特征，在 GFP^高BEC中富集Cyr61和Hes1 。单个 Sox9 ^EGFP+细胞形成的类器官表现出依赖于培养条件的异质存活、生长和 HNF4A 激活，这表明外源性信号传导会影响 BEC 异质性。Yap 是维持胆道类器官中Sox9表达所必需的，但胆汁酸不足以在体内和体外诱导 BEC Yap 活性或Sox9 。Sox9 ^EGFP胆管结扎后仍局限于 BEC 和门静脉周围肝细胞。

结论

我们的数据表明，Sox9 ^EGFP水平提供了 Yap 活性的读数并描绘了 BEC 异质性，为分析肝脏中亚群特异性细胞功能提供了一种工具。