Spatial sorting enables comprehensive characterization of liver zonation

Abstract

The mammalian liver is composed of repeating hexagonal units termed lobules. Spatially resolved single-cell transcriptomics revealed that about half of hepatocyte genes are differentially expressed across the lobule, yet technical limitations impeded reconstructing similar global spatial maps of other hepatocyte features. Here, we show how zonated surface markers can be used to sort hepatocytes from defined lobule zones with high spatial resolution. We apply transcriptomics, miRNA array measurements and mass spectrometry proteomics to reconstruct spatial atlases of multiple zonated features. We demonstrate that protein zonation largely overlaps with mRNA zonation, with the periportal HNF4α as an exception. We identify zonation of miRNAs such as miR-122, and inverse zonation of miRNAs and their hepatocyte target genes, highlighting potential regulation of protein levels through zonated mRNA degradation. Among the targets we find the pericentral Wnt receptors Fzd7 and Fzd8 and the periportal Wnt inhibitors Tcf7l1 and Ctnnbip1. Our approach facilitates reconstructing spatial atlases of multiple cellular features in the liver and other structured tissues. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Liver gene regulatory networks: Contributing factors to nonalcoholic fatty liver disease

“…More recently, the same team has harnessed differential expression of landmark surface proteins to identify different lobule layers by fluorescence‐activated cell sorting (FACS), this time with the intention of characterizing in parallel the transcriptomic and proteomic landscapes of zonated hepatocytes. The authors found more than half of the detected hepatic proteins to be significantly zonated (Ben‐Moshe et al, ) and observed overall recapitulation of the general trends observed previously by mRNA analysis (Halpern et al, ), in which genes associated with bile acid biosynthesis, lipid metabolism and cytochrome P450 metabolism were predominantly pericentral, while gluconeogenesis, oxidative phosphorylation and complement and coagulation cascade genes were preferentially periportal. Interestingly, not all genes showed the same zonation pattern mRNA and protein levels.…”

“…Interestingly, not all genes showed the same zonation pattern mRNA and protein levels. For example, HNF4A, a core hepatic TF that regulates the expression of several other liver TFs and is required for normal liver development and function, was only zonated at protein level, showing higher protein content in periportal hepatocytes (Ben‐Moshe et al, ; Figure ). This zonation at protein level is in line with the role of HNF4A in repressing pericentral genes in periportal regions (Colletti et al, ; Stanulović et al, ), and in activating periportal genes (Brosch et al, ).…”

“…Moreover, mice lacking Dicer, a key element in the processing of miRNAs, display widespread changes in liver zonation (Sekine, Ogawa, McManus, Kanai, & Hebrok, ), indicating that miRNAs are involved in the regulation of zonated gene expression. Accordingly, it has been observed that a large proportion of hepatic miRNAs exhibit zonation in mouse livers, including the most abundant hepatic miRNA miR‐11‐5p (Ben‐Moshe et al, ). miRNAs also seem to be particularly important in the incorporation of Wnt signaling cues to define hepatic zonation, as several key components of the Wnt signaling pathway show clear anti‐correlation with the miRNAs they are regulated by across different lobule regions (Ben‐Moshe et al, ).…”

A Single‐Cell Perspective of the Mammalian Liver in Health and Disease

“…A technical limitation of scRNA‐seq is that it is best suited for quantitative analysis of highly expressed genes. To address this challenge and more comprehensively characterize the molecular nature of liver zonation, Ben‐Moshe et al combined cell sorting using zone‐specific cell surface markers (CD73 and E‐cadherin) and gating strategies to obtain hepatocytes corresponding to different zones . RNA sequencing and proteomic analyses of these pooled hepatocytes revealed greater details of spatial gene expression in the liver at mRNA, micro RNA and protein levels.…”

“…Several principles of metabolic and signaling zonation have emerged from these studies. For example, periportal hepatocytes assume several energetically demanding tasks, such as secretion of plasma proteins, gluconeogenesis, and urea cycle, which are coupled to their access to oxygen‐rich blood supply and high capacity for oxidative metabolism . As such, periportal hepatocytes exhibit abundant expression of albumin and key enzymes including PCK1 (gluconeogenesis), ARG1 (urea cycle), and SDHD (mitochondrial oxidation) to carry out these metabolic functions.…”