Deciphering the zebrafish hepatic duct heterogeneity and cell plasticity using lineage tracing and single-cell transcriptomics

Despite the livers recognized regenerative potential, the role of the hepatic ductal cells (a.k.a. biliary epithelial cells), its heterogeneity, and functionality remain incompletely understood in this process. This study provides a comprehensive examination of the molecular and cellular mechanisms underpinning liver ductal development and liver regeneration in zebrafish, with a spotlight on the functional roles of her family genes in these processes. Using state-of-the-art knock-in zebrafish models and single-cell transcriptomics we reveal the differential expression patterns of the different her genes, of which her2, her6, and her9, were identified as specific molecular signatures for distinguishing different ductal cell types with unique morphology and spatial distribution. Particularly, her9 serves as a pan-ductal marker and shows responsiveness to the synergistic effect of Notch and BMP signaling. By analyzing multiple single-cell RNA-seq datasets, we identify numerous ductal markers which are functional proteins for ductal integrity, and most notably CRISPR mutagenesis demonstrates that her9 is essential for hepatocyte recovery. Using multiple transgenic and knock-in zebrafish lines and genetic fate mapping, we provide a detailed characterization of the ductal remodeling process under development and extreme loss of intra-hepatic duct, highlighting the remarkable ductal cell plasticity. Single-cell transcriptomics of lineage-traced her9-expressing liver ducts in static and regenerative states uncover distinct cell clusters with unique molecular signatures and morphology, reflecting the livers regenerative dynamics and highlight relevant key biological processes that could be leveraged to expedite liver regeneration.