Sox9^EGFPdefines biliary epithelial heterogeneity downstream of Yap activity

Abstract

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.

Single‐Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell–Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice

“…1B‐D)—which aligns with Tulasi’s finding based on SOX9 (SRY [sex determining region Y]‐box 9) immunofluorescent staining, reflecting the heterogeneity of their intrahepatic localization and regeneration capacity. ( 19 )…”

Cellular Homeostasis and Repair in the Biliary Tree

“…22 Interestingly, another recent study demonstrated distinct Sox9 EGFP expression levels at the different anatomical sites of the biliary tree. 31 BECs from different regions (IHBDs, common bile duct, and gallbladder) express regional-specific markers, such as SOX4 expression in intrahepatic BECs, trefoil factor 2 in extrahepatic BECs, and SOX17 in gallbladder BECs. 32 33 Following organoid culture, BECs from different regions exhibited similar transcriptional signatures, suggesting a partial loss of their regionalization.…”