scRNA-seq, snRNA-seq and RNA-seq of mouse hepatocytes during liver maturation

Hepatocytes are the predominant cell type in the liver and execute numerous essential biological functions. However, the crucial events and putative regulators during hepatocyte maturation require in-depth investigation. In this study, we performed single-cell and single-nucleus RNA-seq (scRNA-seq and snRNA-seq) to explore the developmental process of hepatocytes. We defined three maturation stages of postnatal hepatocytes, each of which establishes specific metabolic functions and exhibits distinct strategies for regulating proliferation. Hepatic zonation is gradually formed during hepatocyte maturation. The cells or nuclei of different ploidy exhibit zonation preferences in distribution, and asynchrony in the hepatocyte maturation pathway. In addition, combining gene regulatory network analysis and in vivo genetic manipulation, we identified critical maturation- and zonation-related transcription factors. This study not only delineates comprehensive transcriptomic profiles of hepatocyte maturation, but also presents a paradigm to identify functional genes in the development of hepatocyte maturation and zonation by combining genetic manipulation and measuring coordinates in a single-cell developmental trajectory. To investigate the precise developmental process of hepatocyte maturation, we performed mSTRT scRNA-seq of hepatocytes isolated from the Alb-Cre; Rosa-tdTomato mouse strain at embryonic day (E) 17.5, E19.5, P0, P3, P6, P9, P12, P15, P18, P24, P30 and P60. We also performed mSTRT scRNA-seq of 2N, 4N and 8N hepatocytes at P60 to investigate the heterogeneity of hepatocytes with different ploidy. To explore the maturation process of the nucleus with various ploidy, we isolated nuclei from hepatocytes at P0, P9, P18 and P60 and performed snRNA-seq. To validate the functions of TFs during hepatocyte maturation, we knocked out Nr2f2, Usf1, Xbp1 and Tef in hepatocytes and performed scRNA-seq using the Smart-seq 3 method. To validate that CD324 and CD73 are zonated cell surface markers for PV and CV hepatocytes, we performed bulk-cell RNA-seq to CD324+ cells and CD73+ cells, respectively.