Single-cell activation screen identifies hepatic maturation regulators with zonal resolution

The maturation of lineage-committed embryonic hepatocytes requires both the timed activation of metabolic gene regulatory networks (GRNs) and silencing of embryonic programs to achieve adult hepatic functions. However, in vitro derivation of mature hepatocytes remains imperfect, and key transcriptional regulators governing GRN rewiring during late development are still insufficiently defined. To address this, we generated a developmental reference atlas and employed a dCas9 activation screen with single-cell transcriptomics on primary mouse embryonic hepatocytes, enabling effect ranking among late-onset transcription regulators. We identify Nr1i3 as a potent inducer of pericentrally expressed metabolic genes and Nfix as a critical suppressor of embryonic and periportal signatures. Supplementing liver zonation patterning signals with these regulators further enhanced the expression of pericentrally zonated metabolic genes, emphasizing the importance of a microenvironment-targeted approach. Our screening and analysis therefore highlight regulatory mechanisms underlying organ maturation and offer general strategies for improving the functionality of in vitro-derived cells. Overall design: We generated a hepatocyte transcriptome dataset covering embryonic to adult stages using bulk RNA-seq of purified hepatocytes and scRNA-seq of liver cells. This dataset provided insights into the kinetics of gene expression, enabling the identification of suitable marker genes for assessing hepatic maturation with zonal resolution, as well as 61 transcriptional regulators activated during late development. In addition, we established a scalable, multi-parallel gain-of-function perturbation dataset through pooled single-cell dCas9-activation screening. This experimental validation of late-onset transcriptional regulators allowed us to identify suppressors of embryonic genes and activators of metabolic genes during liver maturation.