Sox9(EGFP) Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity

• Type of Material: Article
• Authors: Deepthi Y. Tulasi, University of North Carolina; Diego Martinez Castaneda, University of North Carolina; Kortney Wager, University of North Carolina; Connor B. Hogan, Emory University; Karel P. Alcedo, University of North Carolina; Jesse R. Raab, University of North Carolina; Adam Gracz, Emory University
• Language: English
• Date: 2021-03-25
• Publisher: Elsevier Inc.
• Publication Version: Final Published Version
• Copyright Statement: © 2021 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.jcmgh.2021.01.009
• Title of Journal or Parent Work: Cellular and molecular Gastroenterology and Hepatology
• Volume: 11
• Issue: 5
• Start Page: 1437
• End Page: 1462
• Grant/Funding Information: The Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.; K.P.A was supported by the National Cancer Institute (T32 CA071341); Supported by the American Gastroenterological Association (Research Scholar Award to A.D.G.), the National Institutes of Diabetes and Digestive and Kidney Diseases (P30 DK34987 to R. Sandler, pilot award to A.D.G.); Emory University Department of Medicine (start-up funds to A.D.G.).
• Keywords: Hepatocytes; Ducts; Functional heterogeneity; Cholangiocytes; SOX9; Gastroenterology & Hepatology; Biliary Epithelium; Sox9; Gene expression; Science & Technology; Growth; Life Sciences & Biomedicine; Liver stem-cells; Long-term expansion; Yap; Mouse liver
• Research Categories: Health Sciences, Oncology; Biology, Cell; Biology, Genetics; Biology, Physiology

Abstract

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 Sox9EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types. Methods Sox9EGFP 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. Sox9EGFP 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. Sox9EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFPhigh BECs. Single Sox9EGFP+ 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. Sox9EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation. Conclusions Our data demonstrate that Sox9EGFP levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.