In Search of Zonation Markers to Identify Liver Functional Disorders

A substantial amount of research is being conducted on zonation markers to identify hepatic injuries and disorders based on the structural and functional zonation of the liver. In contrast to metabolic zonation, hepatocyte ploidy reflects the capability of liver regenerative turnover. Nonetheless, many knowledge gaps remain in the understanding of the links between liver disorders and altered zonation and ploidy, partially owing to the lack of sufficient zonation markers. Under this setting, we recapitulated the currently known and prospective markers used to identify normal and altered liver zonation in different disorders. Furthermore, we discussed new findings from studies that have used advanced methodologies to identify potential markers with greater accuracy. We also elaborated on the perspectives and future applications of zonation research in the early detection of various liver diseases.

The Conundrum of the Pericentral Hepatic Niche: WNT/-Catenin Signaling, Metabolic Zonation, and Many Open Questions

WNT/-catenin signaling promotes stemness, proliferation, and cell fate decisions in various tissue stem cell compartments, which maintain organs with a high turnover of cells (e.g., skin, stomach, and gut). Thus, the -catenin target genes AXIN2 and LGR5 are widely considered as tissue stem cell markers. In contrast, AXIN2 and LGR5 are expressed in pericentral hepatocytes, which do not show overt proliferation during liver homeostasis. Given the low hepatocyte turnover, the liver does not require constant high rates of proliferation, whereas WNT/-catenin signaling is critical for metabolic zonation. Yet, WNT/-catenin pathway upregulation, including AXIN2 and LGR5 induction in hepatocytes throughout the liver, enables hepatocyte regeneration in response to various injuries. In this brief review, I discuss the role of WNT/-catenin signaling in controlling metabolic zonation and the conundrum around pericentral hepatocytes that have been proposed as liver stem cells.

Metabolic and non-metabolic liver zonation is established non-synchronously and requires sinusoidal Wnts

The distribution of complementary metabolic functions in hepatocytes along a portocentral axis is called liver zonation. Endothelial secreted Wnt ligands maintain metabolic zonation in the adult murine liver but whether those ligands are necessary to initiate zonation in the immature liver has been only partially explored. Also, numerous non-metabolic proteins display zonated expression in the adult liver but it is not entirely clear if their localization requires endothelial Wnts. Here we used a novel transgenic mouse model to compare the spatial distribution of zonated non-metabolic proteins with that of typical zonated metabolic enzymes during liver maturation and after acute injury induced by carbon tetrachloride (CCl4). We also investigated how preventing Wnt ligand secretion from endothelial cells affects zonation patterns under homeostasis and after acute injury. Our study demonstrates that metabolic and non-metabolic zonation are established non-synchronously during maturation and regeneration and require multiple endothelial Wnt sources.

In vitro metabolic zonation through oxygen gradient on a chip

Among the multiple metabolic signals involved in the establishment of the hepatic zonation, oxygen could play a key role. Indeed, depending on hepatocyte position in the hepatic lobule, gene expression and metabolism are differently affected by the oxygen gradient present across the lobule. The aim of this study is to understand whether an oxygen gradient, generated in vitro in our developed device, is sufficient to instruct a functional metabolic zonation during the differentiation of human embryonic stem cells (hESCs) from endoderm toward terminally differentiated hepatocytes, thus mimicking the in vivo situation. For this purpose, a microfluidic device was designed for the generation of a stable oxygen gradient. The oxygen gradient was applied to differentiating hESCs at the pre-hepatoblast stage. The definitive endoderm and hepatic endoderm cells were characterized by the expression of the transcription factor SOX-17 and alpha-fetoprotein (AFP). Immature and mature hepatocytes were characterized by hepatocyte nuclear factor 4-alpha (HNF-4α) and albumin (ALB) expression and also analyzed for cytochrome P450 (CYP3A4) zonation and glycogen accumulation through PAS staining. Metabolic zonated genes expression was assessed through quantitative real time PCR. Application of the oxygen gradient during differentiation induced zonated glycogen storage, which was higher in the hepatocytes grown in high pO2 compared to those grown in low pO2. The mRNA levels of glutamine synthetase (GLUL), beta-catenin (CTNNB) and its direct target cyclin D1 (CCND1) showed significantly higher expression in the cells grown in low pO2 compared to those grown in high pO2. On the contrary, carbamoyl-phosphate synthetase 1 (CPS1), ALB, the proliferative marker ki67 (MKI67) and cyclin A (CCNA) resulted to be significantly higher expressed in cells cultured in high pO2 compared to those cultured in low pO2. These results indicate that the oxygen gradient generated in our device can instruct the establishment of a functional metabolic zonation in differentiating hESCs. The possibility to obtain differentiated hepatocytes in vitro may allow in the future to deepen our knowledge about the physiology/pathology of hepatocytes in relation to the oxygen content.