Sox9 EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity

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 transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types. Sox9 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 mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence. BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9 BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFP BECs. Single Sox9 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 remains restricted to BECs and periportal hepatocytes after bile duct ligation. Our data demonstrate that Sox9 levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.