Abstract The conclusive identity of Wnt proteins regulating liver zonation (LZ) and regeneration (LR) remains unclear despite an undisputed role of β-catenin. Using single-cell analysis of liver cells from various species, a conserved Wnt2 and Wnt9b expression in endothelial cells (ECs) in zone 3 shown to be the major Wnt cell source, was identified. Conditional EC-elimination of Wnt2 and Wnt9b led to perturbation of LZ with not only loss of β-catenin targets in zone 3, but also re-appearance of zone 1 genes in zone 3, unraveling dynamicity as revealed by single-cell spatial transcriptomics using Molecular Cartography. Defective LR observed in the knockouts phenocopied other models of defective hepatic Wnt signaling. Administration of a tetravalent antibody to activate Wnt signaling rescued LZ and LR in the knockouts. Molecular Cartography on the livers of the agonist-treated animal revealed changes in LZ. Administration of the agonist also promoted LR in acetaminophen overdose acute liver failure (ALF) fulfilling an unmet clinical need. Overall, we report an unequivocal role of EC-Wnt2 and Wnt9b in LZ and LR and show the role of Wnt activators as regenerative therapy for ALF.

Dynamic control of metabolic zonation and liver repair by endothelial cell Wnt2 and Wnt9b revealed by single cell spatial transcriptomics using Molecular Cartography