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Liver development is controlled by key signals and transcription factors that drive cell proliferation, migration, differentiation and functional maturation. In the adult liver, cell maturity can be perturbed by genetic and environmental factors that disrupt hepatic identity and function. Developmental signals and fetal genetic programmes are often dysregulated or reactivated, leading to dedifferentiation and disease. Here, we highlight signalling pathways and transcriptional regulators that drive liver cell development and primary liver cancers. We also discuss emerging models derived from pluripotent stem cells, 3D organoids and bioengineering for improved studies of signalling pathways in liver cancer and regenerative medicine.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells, and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

Cell-fate determination is influenced by interactions between master transcription factors (TFs) and cis-regulatory elements. Hepatocyte nuclear factor 4 alpha (HNF4A), a liver-enriched TF, acts as a master controller in specification of hepatic progenitor cells by regulating a network of TFs to control onset of hepatocyte cell fate. Using analysis of genome-wide histone modifications, DNA methylation, and hydroxymethylation in mouse hepatocytes, we show that HNF4A occupies active enhancers in hepatocytes and is essential for active histone and DNA signatures, especially acetylation of lysine 27 of histone 3 (H3K27ac) and 5-hydroxymethylcytosine (5hmC). In mice lacking HNF4A protein in hepatocytes, we observed a decrease in both H3K27ac and hydroxymethylation at regions bound by HNF4A. Mechanistically, HNF4A-associated hydroxymethylation (5hmC) requires its interaction with ten-eleven translocation methylcytosine dioxygenase 3 (TET3), a protein responsible for oxidation from 5mC to 5hmC. Furthermore, HNF4A regulates TET3 expression in liver by directly binding to an enhancer region. Conclusion: In conclusion, we identified that HNF4A is required for the active epigenetic state at enhancers that amplifies transcription of genes in hepatocytes.

The Hippo signaling pathway regulates many cellular processes, but has been specifically associated with control organ size and tumor growth. Yes-associated protein 1 (YAP1) is a transcriptional cofactor, in the Hippo pathway, that regulates gene expression when localized in the nucleus. Elevated expression of YAP1 in adult mouse liver leads to hepatomegaly and can cause hepatocellular carcinoma; while the loss of function studies reveal its importance in regulating cholangiocyte development. Here, we report the expression of YAP1 in mouse embryonic and postnatal hepatic cells, using AFP-GFP transgenic mice to identify the hepatocyte lineage. At embryonic day (E) 8.5, YAP1 is highly expressed in the endoderm, but is not present in the nucleus. Between E9.5-12.5, hepatic cells display low levels of nuclear and non-nuclear YAP1. The nuclear expression of YAP1 is first detected in a small subset of hepatic cells starting at E13.5 when the hepatoblasts begin to differentiate into hepatocytes and cholangiocytes. At E18.5, nuclear YAP1 is nearly undetectable in hepatoblasts and hepatocytes, but enriched within the nuclei of cholangiocytes. These levels remain similar postnatally, consistent with the role of YAP1 in cholangiocyte specification and maintenance.