The liver’s remarkable capacity to maintain proper tissue mass after injury has been known since ancient times. However, there has been considerable debate about the source of new liver cells that contribute to tissue growth, maintenance, and regeneration. Multiple studies have reported that disparate cell populations in the liver serve as rare stem cells, whereas others have proposed that most hepatocytes are similar in their regenerative activity regardless of position or function. Although hepatocytes appear histologically homogeneous, the liver lobule is actually organized into concentric zones, or rings, in which hepatocytes express different metabolic enzymes across the portal vein–to–central vein axis through which blood flows. Recently, single-cell profiling has enriched our understanding of the extraordinary diversity of hepatocytes, but this “zonal” heterogeneity has not been functionally interrogated in the context of tissue homeostasis, because the critical genetic labeling tools have not been available.

Previous efforts to identify the most-regenerative hepatocytes have not definitively resolved fundamental questions about whether regenerative activity is spatially restricted within particular zones or whether rare or common subsets of hepatocytes are responsible. This uncertainty was in part because fate mapping had only been performed on a few hepatocyte subsets and without side-by-side comparisons. We sought to systematically address fundamental questions about the source of new liver cells by generating a panel of 11 new CreER knock-in mouse models that label zonal subpopulations across the liver lobule. By using these tools in tandem with three existing CreER lines, tissue maintenance and regeneration as a function of zonal position were assessed.

In contrast to the idea that all hepatocytes across the lobule contribute equally to regeneration, we identified major differences between hepatocytes from different locations. During steady-state homeostasis, zone 1 cells near the portal vein decreased in number over time, as did zone 3 cells near the central vein on the opposite end of the lobule. However, midlobular zone 2 hepatocytes marked by the hepcidin antimicrobial peptide 2 (Hamp2) gene were in large part responsible for homeostatic repopulation. Zone 2 cells were also sheltered from toxic injuries affecting either end of the lobule and thus were well positioned to contribute to regeneration after these insults. To define the mechanistic basis of these lineage-tracing results, single-cell and bulk RNA sequencing transcriptomics were used to define genes that were specifically up- or down-regulated in zone 2. We then used in vivo CRISPR knockout and activation screening to identify functionally important pathways that regulate zone 2 proliferation. These methods revealed that zone 2 repopulation is driven by the insulin-like growth factor binding protein 2–mechanistic target of rapamycin–cyclin D1 (IGFBP2-mTOR-CCND1) axis.

Different regions of the liver lobule exhibit differences in their contributions to hepatocyte turnover, and zone 2 is an important source of new hepatocytes during homeostasis and regeneration. These results challenge the idea that stem cells near the portal or central veins have the highest rates of liver repopulation, but they also support the principle that there are important zonal differences in hepatocyte biology. This study reconciles findings from multiple groups and offers a more unified view of hepatocyte repopulation. The identification of zone 2 hepatocytes as a regenerative population has far-reaching implications for the cellular basis of …