Imbalanced Activation of Wnt-/β-Catenin-Signaling in Liver Endothelium Alters Normal Sinusoidal Differentiation

Endothelial wingless-related integration site (Wnt)-/β-catenin signaling is a key regulator of the tightly sealed blood–brain barrier. In the hepatic vascular niche angiokine-mediated Wnt signaling was recently identified as an important regulator of hepatocyte function, including the determination of final adult liver size, liver regeneration, and metabolic liver zonation. Within the hepatic vasculature, the liver sinusoidal endothelial cells (LSECs) are morphologically unique and functionally specialized microvascular endothelial cells (ECs). Pathological changes of LSECs are involved in chronic liver diseases, hepatocarcinogenesis, and liver metastasis. To comprehensively analyze the effects of endothelial Wnt-/β-catenin signaling in the liver, we used endothelial subtype-specific Clec4g-iCre mice to generate hepatic ECs with overexpression of Ctnnb1. In the resultant Clec4g-iCretg/wt;Ctnnb1(Ex3)fl/wt (Ctnnb1OE−EC) mice, activation of endothelial Wnt-/β-catenin signaling resulted in sinusoidal transdifferentiation with disturbed endothelial zonation, that is, loss of midzonal LSEC marker lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve1) and enrichment of continuous EC genes, such as cluster of differentiation (CD)34 and Apln. Notably, gene set enrichment analysis revealed overrepresentation of brain endothelial transcripts. Activation of endothelial Wnt-/β-catenin signaling did not induce liver fibrosis or alter metabolic liver zonation, but Ctnnb1OE−EC mice exhibited significantly increased plasma triglyceride concentrations, while liver lipid content was slightly reduced. Ctnnb1 overexpression in arterial ECs of the heart has been reported previously to cause cardiomyopathy. As Clec4g-iCre is active in a subset of cardiac ECs, it was not unexpected that Ctnnb1OE−EC mice showed reduced overall survival and cardiac dysfunction. Altogether, balanced endothelial Wnt-/β-catenin signaling in the liver is required for normal LSEC differentiation and for maintenance of normal plasma triglyceride levels.

Liver Zonation – Revisiting Old Questions With New Technologies

Despite the ever-increasing prevalence of non-alcoholic fatty liver disease (NAFLD), the etiology and pathogenesis remain poorly understood. This is due, in part, to the liver’s complex physiology and architecture. The liver maintains glucose and lipid homeostasis by coordinating numerous metabolic processes with great efficiency. This is made possible by the spatial compartmentalization of metabolic pathways a phenomenon known as liver zonation. Despite the importance of zonation to normal liver function, it is unresolved if and how perturbations to liver zonation can drive hepatic pathophysiology and NAFLD development. While hepatocyte heterogeneity has been identified over a century ago, its examination had been severely hindered due to technological limitations. Recent advances in single cell analysis and imaging technologies now permit further characterization of cells across the liver lobule. This review summarizes the advances in examining liver zonation and elucidating its regulatory role in liver physiology and pathology. Understanding the spatial organization of metabolism is vital to further our knowledge of liver disease and to provide targeted therapeutic avenues.

Single-nucleus RNA-seq2 reveals functional crosstalk between liver zonation and ploidy

Single-cell RNA-seq reveals the role of pathogenic cell populations in development and progression of chronic diseases. In order to expand our knowledge on cellular heterogeneity, we have developed a single-nucleus RNA-seq2 method tailored for the comprehensive analysis of the nuclear transcriptome from frozen tissues, allowing the dissection of all cell types present in the liver, regardless of cell size or cellular fragility. We use this approach to characterize the transcriptional profile of individual hepatocytes with different levels of ploidy, and have discovered that ploidy states are associated with different metabolic potential, and gene expression in tetraploid mononucleated hepatocytes is conditioned by their position within the hepatic lobule. Our work reveals a remarkable crosstalk between gene dosage and spatial distribution of hepatocytes.

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.

“Single-nucleus RNA-seq2 reveals a functional crosstalk between liver zonation and ploidy”

Single-cell RNA-seq reveals the role of pathogenic cell populations in development and progression of chronic diseases. In order to expand our knowledge on cellular heterogeneity we have developed a single-nucleus RNA-seq2 method that allows deep characterization of nuclei isolated from frozen archived tissues. We have used this approach to characterize the transcriptional profile of individual hepatocytes with different levels of ploidy, and have discovered that gene expression in tetraploid mononucleated hepatocytes is conditioned by their position within the hepatic lobe. Our work has revealed a remarkable crosstalk between gene dosage and spatial distribution of hepatocytes.